Rbp1l1, a novel retinoblastoma binding protein-related gene encoding an antigenic epitope and methods of using thereof

ABSTRACT

Disclosed herein is a novel retinoblastoma binding protein-related gene that encodes an antigenic epitope, IKP-SLGSKK, which is differentially expressed in cancers, such as breast, lung, colon, pancreas, and ovarian cancer. Polynucleotides that encode and polypeptides that comprise the antigenic epitope and variants thereof are disclosed. Also, disclosed are methods of using the polynucleotides and polypeptides of the present invention in immunotherapies and immunnassays.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/275,518 filed 14 Mar. 2001, which names Jia-ning Caoas the inventor and is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant Nos.CA 12582, CA 56059, and CA 13579 awarded by National Institutes ofHealth/National Cancer Institute.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates generally to methods of detecting ordiagnosing a plurality of different cancers. In particular, the presentinvention relates to a retinoblastoma binding protein-related gene thatis differentially expressed in cancer cells as compared to normal cells.

[0005] 2. Description of the Related Art

[0006] Breast, lung, colon, pancreas, and ovarian cancers are aggressiveforms of cancer, difficult to diagnose, or both. In order to detectbreast cancer by physical examination, a breast tumor must be of a largeenough size and mammography is limited by the skill of the observer, thequality of the mammogram, often result in over 80% false positives andover 10% false negatives. See Wright, et al. (1995) Lancet 346:29-32.Generally, there are no observable clinical symptoms of lung canceruntil the advanced stage. In fact, only 16% of lung cancers arediscovered before the disease has spread. Likewise, colon cancer isdifficult to diagnose as clinical symptoms such as rectal bleeding,pain, abdominal distension, or weight loss are observable only after thedisease is advanced and not surgically curable. Fecal occult blood testsare difficult to conduct due to lack of compliance, are poorlysensitive, exhibit little predicative value, and often lead tocolonoscopic examination which is an expensive and invasive procedure.Pancreatic cancer produces few specific symptoms in its early stages andusually is detected at an advanced and incurable stage. Thus, pancreaticcancer exhibits the lowest survival rate of any major cancer.Specifically, the average survival after diagnosis is less than 6 monthsand fewer than 5% of patients survive 5 years. See Rosenberg, L. (1997)Pancreatol. 22:81-93; and Flanders and Foulkes (1996) J. Med. Genet.33:889-898). Likewise, ovarian cancer is difficult to diagnose andeffective treatment depends on early detection.

[0007] Molecular identification of tumor antigens that can elicit bothantibody and cellular immune responses in humans has been an importantfocus in the development of therapeutic methods that are active andspecific against various cancers and tumors. Many strategies have beenused to characterize and detect tumor antigens. See e.g. DePlaen et al.(1988) PNAS USA 85:2275-2280; Mandelboim, et al. (1994) Nature 369:69;and Sahin, et al. (1995) PNAS USA 92:11810-11913.

[0008] Numerous human tumor antigens have been identified. See e.g., Vander Bruggen, et al. (1991) Science 254:1643-1647 (MAGE-1); Brichard, etal. (1993) J. Exp. Med. 178:489-495; Coulie, et al. (1994) J. Exp. Med.180:35-42; Kawakami, et al. (1994) PNAS USA 91:3515-3519; Clark, et al.(1994) Nature Genetics 7:502-508 (SSX-2); Crew, et al. (1995) EMBO J.144:2333-2340 (SSX-1); DeLeeuw, et al. (1996) Cytogenet. Genet.73:179-183 (SSX-3); Gure, et al. (1997) Int. J. Cancer, 72:965-971(SSX4); Meuwissen, et al. (1992) EMBO J. 11(13):5091-5100 (SCP-1);Tureci, et al. (1996) Cancer Res. 56:4766-4772 (HOM-MEL-40); Chen, etal. (1997) PNAS USA 94:1914-1918 (NY-ESO-1); Boel, et al. (1995)Immunity 2:167-175 (BAGE); and U.S. Pat. Nos. 5,610,013 and 5,648,226(GAGE). Many of these tumor antigens have been the focus of cancerimmunotherapy. Unfortunately, for a variety of reasons, many of thetumor antigens known in the art are not useful in cancer immunotherapy.

[0009] Thus, a need still exists for effective and accurate cancerdiagnostics and therapies.

SUMMARY OF THE INVENTION

[0010] The present invention generally relates to a retinoblastomabinding protein-related gene encoding an antigenic epitope and methodsof using thereof.

[0011] In some embodiments, the present invention relates to aretinoblastoma binding protein-related gene that encodes a polypeptidecomprising SEQ ID NO:3 or a variant thereof The variant may specificallybind to an antibody raised against the polypeptide comprising SEQ IDNO:3 or an antibody obtained from a subject having cancer, such asbreast, lung, colon, pancreas, or ovarian cancer. In some embodiments,the polynucleotide comprises a SEQ ID NO:1 or a fragment or variantthereof. In other embodiments, the polynucleotide consists of SEQ IDNO:1 or a fragment thereof. In some embodiments, the polypeptidecomprises SEQ ID NO:2 or a fragment or variant thereof. In otherembodiments, the polypeptide consists of SEQ ID NO:2 or SEQ ID NO:3. Insome embodiments, the polynucleotide sequence is at least about 80%identical to SEQ ID NO:1. In some embodiments, the polynucleotidesequence is at least about 90% identical to SEQ ID NO:1. In someembodiments, the polypeptide is at least about 80% identical to SEQ IDNO:2 or SEQ ID NO:3. In some embodiments, the polypeptide is at leastabout 90% identical to SEQ ID NO:2 or SEQ ID NO:3.

[0012] In some embodiments, the present invention relates to apolypeptide encoded by the polynucleotide of the present invention. Insome embodiments, the polypeptide comprises SEQ ID NO:2 or SEQ ID NO:3.In other embodiments, the polypeptide consists of SEQ ID NO:2 or SEQ IDNO:3. In some embodiments, the polypeptide is at least about 80%identical to SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, thepolypeptide is at least about 90% identical to SEQ ID NO:2 or SEQ IDNO:3.

[0013] In some embodiments, the present invention relates to a vectorcomprising a polynucleotide of the present invention.

[0014] In some embodiments, the present invention relates to a hostcomprising a polynucleotide of the present invention.

[0015] In some embodiments, the present invention relates to a fusionprotein comprising a polypeptide of the present invention.

[0016] In some embodiments, the present invention relates to an antibodyor fragment thereof that specifically binds a polypeptide of the presentinvention. In some embodiments, the antibody was raised against apolypeptide comprising SEQ ID NO:2 or SEQ ID NO:3.

[0017] In some embodiments, the present invention relates to apharmaceutical composition comprising a polypeptide or an antibody orfragment of the present invention and a pharmaceutically acceptablevehicle.

[0018] In some embodiments, the present invention relates to animmunogenic composition comprising a polypeptide of the presentinvention.

[0019] In some embodiments, the present invention relates to a vaccinecomprising a polypeptide of the present invention. In preferredembodiments, the vaccine is effective for treating or preventing cancer,such as breast, lung, colon, pancreas, or ovarian cancer.

[0020] In some embodiments, the present invention relates to a method ofinducing an immune response against a cancer or a tumor in a subjectwhich comprises administering to the subject at least one polypeptide ofthe present invention. The cancer may be breast, lung, colon, pancreas,or ovarian cancer.

[0021] In some embodiments, the present invention relates to a method oftreating, preventing, or inhibiting a cancer or a tumor in a subjectwhich comprises administering to the subject at least one polypeptide ofthe present invention, at least one antibody raised against apolypeptide of the present invention, or both. In preferred embodiments,the cancer is breast, lung, colon, pancreas, or ovarian cancer.

[0022] In some embodiments, the present invention relates to a markerfor diagnosing a cancer or susceptibility of obtaining the cancer in asubject comprising a polynucleotide of the present invention. Inpreferred embodiments, the cancer is breast, lung, colon, pancreas, orovarian cancer.

[0023] In some embodiments, the present invention relates to a markerfor diagnosing a cancer or susceptibility of obtaining the cancer in asubject comprising a polypeptide of the present invention.

[0024] In some embodiments, the present invention relates to a methodfor detecting the presence of a cancer in a subject which comprisesobtaining a biological sample from the subject; contacting thebiological sample with a binding agent that binds to a polypeptide ofthe present invention; detecting in the sample an amount of polypeptidethat binds to the binding agent; and comparing the amount of polypeptideto a control and therefrom determining the presence of a cancer in thesubject.

[0025] In some embodiments, the present invention relates to a methodfor detecting the presence of a cancer in a subject which comprisesobtaining a biological sample from the patient; contacting thebiological sample with a polynucleotide of the present invention;detecting in the sample an amount of a second polynucleotide thathybridizes to the polynucleotide; and comparing the amount of the secondpolynucleotide that hybridizes to the polynucleotide to a control, andtherefrom determining the presence of the cancer in the patient.

[0026] In some embodiments, the present invention relates to adiagnostic kit comprising at least one reagent selection from the groupconsisting of: a polynucleotide that encodes a polypeptide comprisingSEQ ID NO:3 or a variant thereof; the polypeptide comprising SEQ ID NO:3or a variant thereof; a polypeptide comprising SEQ ID NO:2 or a fragmentor variant thereof; or an antibody that specifically binds thepolypeptide of the present invention; and instructions for use.

[0027] In some embodiments, the present invention relates to a methodfor inhibiting the development of a cancer in a subject which comprisesincubating CD4⁺ T cells, CD8⁺ T cells, or both with at least onecomponent selected from the group consisting of: at least onepolynucleotide that encodes a polypeptide comprising SEQ ID NO:3 or avariant thereof; the polypeptide comprising SEQ ID NO:3 or a variantthereof; at least one polypeptide comprising SEQ ID NO:2 or a fragmentor variant thereof; and antigen presenting cells that express thepolypeptide of the present invention, such that T cell proliferate; andadministering to the subject an effective amount of the proliferated Tcells, and thereby inhibiting the development of a cancer in thesubject.

[0028] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are intended to provide further explanation of theinvention as claimed. The accompanying drawings are included to providea further understanding of the invention and are incorporated in andconstitute part of this specification, illustrate several embodiments ofthe invention, and together with the description serve to explain theprinciples of the invention.

DESCRIPTION OF THE DRAWINGS

[0029] This invention is further understood by reference to the drawingswherein:

[0030]FIG. 1A illustrates the expression of immunopositive fusionproteins in Escherichia coli and is depicted in sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by Coomassiebrilliant blue staining. Lane a=lysate of isopropylβ-D-thiogalactopyranoside (IPTG)-induced lysogen from clone 131; laneb=lysate of IPTG-induced lysogen from clone 151. The band at 128 kDarepresents the fusion protein containing a 107-amino acid insert.Molecular mass markers (kDa) are indicated to the right.

[0031]FIG. 1B illustrates the expression of immunopositive fusionproteins in Escherichia coli and is depicted in sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by Western blotanalysis with the purified human IgG. Lane a=lysate of isopropylβ-D-thiogalactopyranoside (IPTG)-induced lysogen from clone 131; laneb=lysate of IPTG-induced lysogen from clone 151. The band at 128 kDarepresents the fusion protein containing a 107-amino acid insert.Molecular mass markers (kDa) are indicated to the right.

[0032]FIG. 2 shows the complete nucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence of RBP1L1 gene (SEQ ID NO:2). The aminoacids in the epitope are underlined (SEQ ID NO:3).

[0033]FIG. 3 is an alignment of the sequences of the RBP1L1 protein andthe RBP1 protein (SEQ ID NO:4). The dotted line indicates absentsequence. The amino acids in the epitopes are underlined.

[0034]FIG. 4A provides the Northern blot analysis of RBP1L1 MnRNAexpression in 13 normal human tissues.

[0035]FIG. 4B shows the differential expression of the RBP1L1 gene. The983-bp band is the G3PDH PCR product. The 441-bp band is the RBP1L1 PCRproduct.

[0036]FIG. 5 shows the concentration of specific IgG purified from theserum of a patient with breast cancer that binds to various syntheticpeptides in an enzyme-linked immunosorbent assay (ELISA).

[0037]FIG. 6A shows the immunoperoxidase staining of MCF-7 breast cancercells in the cytoplasm.

[0038]FIG. 6B shows no staining of MCF-7 cells with irrelevant human IgGmonoclonal antibody alone.

[0039]FIG. 6C shows only membrane staining in human PBMCs with thepurified RBP1L1 specific IgG.

[0040]FIG. 6D shows membrane staining of PBMCs with only the secondantibody. Scale bars=100 μm.

[0041]FIG. 7 shows a mRNA microarray that shows that RBP1L1 isdifferentially expressed in cancer cells as compared to normal cells.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention provides a retinoblastoma-bindingprotein-1-like protein-1 (RBP1L1), a novel polypeptide that isdifferentially expressed in breast, lung, colon, pancreas, and ovariancancer cells as compared to normal cells.

[0043] “RBP1L1” denotes a polypeptide encoded by aretinoblastoma-binding protein-1-like protein-1 gene having apolynucleotide sequence (1) set forth in SEQ ID NO:1, (2) hassubstantial identity to the sequence set forth in SEQ ID NO:1, (3) thatencodes the polypeptide of SEQ ID NO:2 or SEQ ID NO:3, (4) that encodesa polypeptide having substantial identity to the sequence of SEQ ID NO:2or SEQ ID NO:3, and (5) variants thereof. RBP1L1 is differentiallyexpressed in cancer cells, such as breast, lung, colon, pancreas, andovarian cancer cells, at a level that that is greater than the level ofexpression in a normal tissue, as determined by conventional methodsknown in the art. RBP1L1 may be obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0044] In a previous study, a tumor-associated antigen corresponding tothe retinoblastoma binding protein-1 (RBP1) was identified by using anallogeneic IgG antibody prepared from the serum of a patient with breastcancer to screen a complementary DNA (cDNA) expression library of MCF-7breast cancer cells. See Cao, J, et al. (1999) Breast Cancer Res. Treat.53:279-290, which is herein incorporated by reference. Although the RBP1gene was present in normal human cells, as well as cancer cells,expression of the antigenic epitope was restricted to certain types ofcancer, such as breast, prostate, and renal cancers. See Fattaey, A. R.,et al. (1993) Oncogene 8:3149-3146, which is herein incorporated byreference.

[0045] In humans, RBP1 contains a heptameric antibody-binding peptideepitope, KASIFLK (SEQ ID NO:5), corresponding to RBP1 amino acids250-256 and two overlapping decameric peptide epitopes, GLQKASIFLK (SEQID NO:6) and KASIFLKTRV (SEQ ID NO:7), corresponding to RBP1 amino acids247-256 and 250-259, respectively. These epitopes were highlyimmunogenic and induced both specific antibody and cytotoxicT-lymphocyte (CTL) responses. CTL responses induced by the decamericpeptide epitopes exhibited strong cytotoxic activity against HLA-A2- andHLA-A3-positive breast cancer cells. See Takahashi, T., et al. (1999)Br. J. Cancer 81:342-349, which is herein incorporated by reference. Theinvestigation of the KASIFLK epitope (SEQ ID NO:5) led to the discoveryof the present invention—the RBP1L1 polynucleotide that encodes apolypeptide comprising an antigenic nonameric peptide epitope, IKPSLGSKK(SEQ ID NO:3).

[0046] As disclosed herein, the RBP1L1 gene was isolated from a MCF-7cDNA expression library screened with purified human IgG from a patientwith breast cancer. The complete 5802 bp RBP1L1 cDNA encodes a 1226amino acid polypeptide comprising the epitope, IKPSLGSKK (SEQ ID NO:3).The polypeptide sequence of RBP1L1 shares 74% amino acid identity withthat of the partial cDNA sequence of the retinoblastoma-binding proteinand 37% identity with retinoblastoma binding protein-1 (RBP1), whichreportedly suppress cell growth by interacting with the retinoblastomafamily members and histone deacetylases to repress transcriptionalactivity.

[0047] Little or no expression of RBP1L1 transcripts was detected innormal adult pancreas, prostate, ovary, adrenal medulla, thyroid,adrenal cortex, spleen, thymus, colon, stomach, and peripheral bloodmononuclear cells (PBMCs), although expression was detected in normaltestis. Consistent with this observation, RT-PCR analysis of normaltissues did not detect RBP1L1 mRNA, except in testis. However, in humanbreast, lung, colon, ovaries, and pancreas cancers, high levels ofRBP1L1 mRNA were detected. Thus, RBP1L1 mRNA is expressed abundantly incancer cells and in normal testicular cells. This restricted pattern ofexpression or differential expression pattern may be used to diagnose abroad range of human cancers.

[0048] As provided in the Examples below, purified human IgG from anindividual having breast cancer binds to a nonameric peptide antigen,IKPSLGSKK (SEQ ID NO:3), encoded by the RBP1L1 polypeptide.Immunohistochemical and cytological studies with this antibody revealthat the antigen is located in the cytoplasm. Because of the extensivesequence conservation between RBP1L1 and RBP1 (64% identity in theN-terminal 450 amino acids and 42% identity in C-terminal 300 aminoacids) it is likely that the functional pathway of RBP1L1 in partmirrors that of RBP1. Like RBP1, RBP1L1 also comprises an ARID and aBRIGHT DNA binding domain. Both antigens, IKPSLGSKK (SEQ ID NO:3) andKASIFLK (SEQ ID NO:5) are over-expressed in human cancer cells that aredetected by human antibodies. The cellular localization of both antigensis localized to present mainly in the cytoplasm of cancer cells.

[0049] Previous studies show that serologically identified antigens arerecognized by HLA class I-restricted CTLs and can induce antibodyresponses in subjects with tumors. See Morioka, N., et al. (1994) J.Immunol. 153:5650-5658; Takahashi, T., et al. (1997) Cell Immunol.178:162-171; and Chen, Y. T., et al. (1997) PNAS USA 94:1914-1918, whichare herein incorporated by reference. Since the antigenic peptideKASIFLK (RBP1 amino acids 250-256) (SEQ ID NO:5) is serologicallysimilar to the antigenic peptide of the present invention, IKPSLGSKK(SEQ ID NO:3), it is expected that in vitro stimulation of HLA-A2- andHLA-A3-positive PBMCs with peptides comprising the IKPSLGSKK antigen(SEQ ID NO:3) will generate peptide-specific CTLs that are highlycytotoxic to HLA-A2- and HLA-A3-positive cancer cells but not to normalcells. See Chen, J. L., et al. (2000) J. Immunol. 165:948-955; andTakahashi, T., et al. (1999) Br. J. Cancer 81:342-349, which are hereinincorporated by reference. Therefore, the IKPSLGSKK antigenic epitope(SEQ ID NO:3) of the present invention may be used as a target forcancer cell destruction via a dual-effector immune system.

[0050] Since MAGE, TRP-2, and gp100 tumor-associated antigens have beenshown to elicit strong antibody responses to their respectiverecombinant antigens in melanoma patients immunized with anantigen-containing melanoma cell vaccine, the present invention providesan antigenic epitope, IKPSLGSKK (SEQ ID NO:3), which may be used inimmunotherapies for treating cancer.

[0051] Because of the protein sequence identity and similarimmunological features between RBP1L1 and RBP1, RBP1L1 may associatewith the pRB-pocket and regulate the transcription of genes that controlthe cell cycle, differentiation, proliferation, and apoptosis.Specifically, since (1) RBP1 binds to the pocket of pRB, (2) RBP1represses transcriptional activity by interacting with p130-E2F andpRB-E2F complexes during cell cycle arrest, (3) RBP1 repressesE2F-dependent transcription by recruitment of histone deacetylasescomplex, and (4) over-expression of RBP1 inhibits E2F-dependent geneexpression and suppresses cell growth (Lai, A., et al. (1999) Oncogene18:2091-2100; Dyson, N. (1998) Genes Dev. 12:2245-2262; and Lai, A., etal. (2001) Mol. Cell. Biol. 21(8):2918-2932), RBP1L1 may also bind thepocket of pRB, repress transcriptional activity, or affect the cellcycle and growth of a cell, or a combination thereof.

[0052] Genes with high mRNA expression in human testis and tumor cellsare not unique and mRNAs of many of the genes for humancancer-associated antigens, identified by using human antibodies toscreen recombinant cDNA expression libraries, are also overexpressed inthe testis. See Chen, Y. T., et al. (1997) PNAS USA 94:1914-1918; Vander Bruggen, P., et al. (1991) Science 254:1643-1647; and Gure, A. O.,et al. (2000) Int. J. Cancer 85:726-732, which are herein incorporatedby reference. The present invention provides the first retinoblastomabinding-related protein gene that encodes a cancer-associated antigenand is highly expressed by various cancer types (and normal testicularcells). The RBP1L1 polynucleotide sequence shows no homology to anyreported cancer/testis genes.

[0053] The high expression of RBP1L1 in normal testicular cells, doesnot diminish the use of RBP1L1 in immunotherapy because the testis is animmunologically privileged site. Thus, the testicular cells expressingthe tumor antigen will escape direct contact by antigen-presentingcells, and CTLs and antibody responses induced by peptide antigensderived from cancer/testis antigen genes may attack autologous cancercells without killing normal testicular cells. The lack of HLA class Iexpression on the surface of testicular cells also favors their abilityto escape from CTL recognition and killing. Thus, the present inventionprovides methods of using the antigenic epitope, IKPSLGSKK (SEQ IDNO:3), or a peptide containing this epitope, in cancer and tumorimmunotherapy.

[0054] The present invention is generally directed to compositions andmethods for using the compositions in the diagnosis and treatment ofcancer, such as breast, lung, colon, pancreas, and ovarian cancer. Thecompositions of the present invention include RBP1L1 polypeptides,polynucleotides encoding such polypeptides, binding agents such asantibodies, antigen presenting cells (APCs), immune system cells (e.g.,T cells), and combinations thereof.

[0055] The present invention provides RBP1L1 polynucleotides andvariants thereof. In preferred embodiments the polynucleotides areisolated. As used herein, “isolated” polynucleotide refers to a DNAmolecule that is isolated from its native environment. An “isolated”polynucleotide may be substantially isolated away from, or purified freefrom, total genomic DNA of the species from which the DNA segment isobtained. An “isolated” polynucleotide may include a DNA segment that isseparated from other DNA segments with which is normally or nativelyassociated at either the 5′ end, 3′ end, or both. An “isolated”polynucleotide may include a DNA segment that is substantially away fromother coding sequences, and does not contain large portions of unrelatedcoding DNA, such as large chromosomal fragments or other functionalgenes or polypeptide coding regions. “Nucleic acid sequence”, “nucleicacid molecule”, and “polynucleotide” are used interchangeably to referto an oligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin andmay be single- or double-stranded, and represent the sense or antisensestrand.

[0056] The polynucleotides of the present invention may include genomicsequences, extra-genomic and plasmid-encoded sequences and smallerengineered gene segments that express, or may be adapted to express,proteins, polypeptides, peptides, and the like. The polynucleotides ofthe present invention may be in its native form or syntheticallymodified. The polynucleotides of the present invention may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includemRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mMRNA molecules, which do not containintrons. Additional coding or non-coding sequences may be present withina polynucleotide of the present invention, and a polynucleotide may belinked to other molecules, support materials, or both.

[0057] The polynucleotides of the present invention may comprise anative sequence, i.e., an endogenous sequence that encodes the RBP1L1polypeptide or a portion thereof, or may comprise a variant or abiological or antigenic functional equivalent thereof. Polynucleotidevariants may contain one or more substitutions, additions, deletions,insertions, or combinations thereof so long as the biological activity,such as immunogenicity, of the encoded polypeptide is not diminished,relative to the RBP1L1 polypeptide. A “variant” of a polynucleotiderefers to the chemical modification of a nucleic acid encoding RBP1L1 orthe encoded RBP1L1. Illustrative of such modifications would bereplacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid variant encodes a polypeptide that retains essential biologicalcharacteristics of the natural molecule.

[0058] The term “biologically active”, as used herein, refers to apolypeptide or polynucleotide having structural, regulatory, orbiochemical functions of a naturally occurring molecule.

[0059] The polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol.

[0060] In other embodiments, the present invention is directed topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art.

[0061] As used herein, “stringent conditions” refers to the “stringency”which occurs within a range from about 5° C. below the meltingtemperature (Tm) of the probe to about 20° C. to about 25° C. below Tm.As will be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences. For purposes of illustration, suitablemoderately stringent conditions for testing the hybridization of apolynucleotide of this invention with other polynucleotides includeprewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);hybridizing at about 50° C. to about 65° C., 5×SSC, overnight; followedby washing twice at about 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS.

[0062] Moreover, it will be appreciated by those of ordinary skill inthe art that, as a result of the degeneracy of the genetic code, thereare many nucleotide sequences that encode a polypeptide as describedherein. Some of these polynucleotides bear minimal homology to thenucleotide sequence of any native gene. Nonetheless, polynucleotidesthat vary due to differences in codon usage are specificallycontemplated by the present invention. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

[0063] Alternatively, the polypeptides of the present invention may bemade by conventional recombinant DNA techniques such as those disclosedin the Examples below. Thus, the present invention providespolynucleotides that encode the polypeptides of the present invention.In preferred embodiments, the polynucleotides are isolated.

[0064] A polynucleotide that encodes a polypeptide having substantialidentity to either SEQ ID NO:2 or SEQ ID NO:3 can be made by introducingone or more nucleotide substitutions, insertions, or deletions into thenucleotide sequence that encodes SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3 such that one or more amino acid substitutions, insertions, ordeletions are introduced into the encoded polypeptide. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis.

[0065] In other embodiments of the present invention, the polynucleotidesequences provided herein can be used as probes or primers for nucleicacid hybridization. As such, nucleic acid segments that comprise asequence region of at least about a 15 nucleotide long contiguoussequence that has the same sequence as, or is complementary to, a 15nucleotide long contiguous sequence disclosed herein are contemplated.Longer contiguous identical or complementary sequences up to full-lengthsequences are also contemplated.

[0066] The ability of such nucleic acid probes to specifically hybridizeto a sequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

[0067] Polynucleotides having sequence regions consisting of contiguousnucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200nucleotides or so (including intermediate lengths as well), identical orcomplementary to SEQ ID NO:1 or SEQ ID NO:15, are particularlycontemplated as hybridization probes in recombinant DNA and molecularbiology techniques. The total size of fragment, as well as the size ofthe complementary sequence, will ultimately depend on the intended useor application of the particular nucleic acid segment. Smaller fragmentswill generally find use in hybridization embodiments, wherein the lengthof the contiguous complementary region may be varied, such as betweenabout 15 and about 100 nucleotides, but larger contiguouscomplementarily stretches may be used.

[0068] Small polynucleotide fragments of the present invention may bereadily prepared by conventional methods known in the art, for example,directly synthesizing the fragment with an automated oligonucleotidesynthesizer, PCR technology, and recombinant DNA techniques.

[0069] The polynucleotides of the present invention may be identified,prepared, or manipulated by conventional methods known in the art. Forexample, a polynucleotide may be identified by screening a microarray ofcDNAs for tumor-associated expression, i.e., differential expression ascompared to the expression in normal tissue, as determined byconventional methods such as that provided in the Examples herein. Seee.g. Schena et al. (1996) PNAS USA 93:10614-10619, and Heller et al.(1997) PNAS USA 94:2150-2155, which are herein incorporated byreference. Alternatively, the polynucleotides of the present inventionmay be amplified from cDNA prepared from cells expressing the proteinsdescribed herein, such as breast tumor cells.

[0070] Polypeptides and polypeptide fragments of the present inventioncomprise at least one immunogenic portion of RBP1L1 or a variantthereof. “Amino acid sequence”, “amino acid molecule”, “polypeptide”,“protein”, and “peptide”, are used interchangeably to refer to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0071] RBP1L1 polypeptides will react with antibodies raised against apolypeptide comprising SEQ ID No: 2 or SEQ ID NO:3 and may reactdetectably within an immunoassay (such as an ELISA) with antisera from apatient with having breast, lung, colon, pancreas, or ovarian cancer.Polypeptides of the present invention may be of any length. Additionalsequences derived from the native protein and/or heterologous sequencesmay be present, and such sequences may possess further immunogenic orantigenic properties.

[0072] As used herein an “immunogenic portion” is a portion of a proteinthat is recognized by a B-cell and/or T-cell surface antigen receptor.Such immunogenic portions include the antigenic epitope, IKPSLGSKK (SEQID NO:3), or variants thereof. Variants of IKPSLGSKK are polypeptidesthat exhibit a similar sequence homology and bioactivity(immunogenicity) to IKPSLGSKK (SEQ ID NO:3). Immunogenic portions maygenerally be identified using well-known techniques. See Paul,Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) andreferences cited therein, which are herein incorporated by reference.The terms “antigenic determinant” or “antigenic epitope” or “epitope”are used interchangeably to refer to a region of a molecule with theability or potential to elicit, and combine with, an antibody orfragment thereof.

[0073] The polypeptides of the present invention need not be identicalto those exemplified herein so long as the subject polypeptides are ableto induce an immune response against the antigenic epitope orimmunogenic portion of the present invention. Thus, as used herein“variants” of the polypeptides of the present invention refer topolypeptides having insignificant changes. “Insignificant changes” referto modifications in the amino acid sequence of a given polypeptide thatdo not change the biological activity, such as the immunologicalactivity, of the polypeptide. Such insignificant changes include amethionine as the first amino acid residue at the amino terminus,conservative amino acid substitutions, deletions, or insertions, andco-translational or post-translational surface modifications such as theaddition of covalently attached sugars, lipids, or combinations thereof.“Immunologically activity” refers to the capability of the natural,recombinant, or synthetic RBP1L1, or any oligopeptide thereof, to inducea specific immune response in appropriate animals or cells and to bindwith specific antibodies.

[0074] A polypeptide “variant” of the present invention also includes apolypeptide that differs from RBP1L1 (SEQ ID NO:2) by one or moresubstitutions, deletions, additions, insertions, or a combinationthereof such that the immunogenicity of the polypeptide is notsubstantially diminished. For example, the ability of a variant to reactwith antibodies specific for IKPSLGSKK (SEQ ID NO:3) may be enhanced orunchanged, relative to the native protein, or may be diminished by lessthan 50%, and preferably less than 20%, relative to the native protein.

[0075] As used herein, a “variant” of RBP1L1 a polypeptide is altered byone or more amino acids. The variant may have “conservative” changes,wherein a substituted amino acid has similar structural or chemicalproperties, e.g. replacement of leucine with isoleucine. More rarely, avariant may have “nonconservative” changes, e.g., replacement of aglycine with a tryptophan. Similar minor variations may also includeamino acid deletions, insertions, or both. Guidance in determining whichamino acid residues may be substituted, inserted, or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, LASERGENEsoftware. Polypeptide variants encompassed by the present inventioninclude those exhibiting at least about 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more identity to the polypeptidesdisclosed herein.

[0076] The polypeptides of the present invention may also be modified toprovide a variety of desired attributes, e.g., improved pharmacologicalcharacteristics, while increasing or at least retaining substantiallyall of the immunological activity of the antigenic epitope. By usingconventional methods in the art, one of ordinary skill will be readilyable to make a variety of polypeptides having substantial identity tothose explicitly provided for herein, and then screen the polypeptidesfor stability, toxicity, and immunogenicity according to the presentinvention.

[0077] Additionally, single amino acid substitutions, deletions, orinsertions can be used to determine which residues are relativelyinsensitive to modification. Amino acid substitutions are preferablymade between relatively neutral moieties, such as alanine, glycine,proline, and the like. Substitutions with different amino acids, ofeither D or L isomeric forms, or amino acid mimetics can be made. Thenumber and types of substitutions, deletions, and insertions depend onthe functional attributes that are sought such as hydrophobicity,immunogenicity, three-dimensional structure, and the like.

[0078] An “amino acid mimetic” as used herein refers to a moiety otherthan a naturally occurring amino acid residue that conformationally andfunctionally serves as a suitable substitute for an amino acid residuein a polypeptide of the present invention. A moiety is a suitablesubstitute for an amino acid residue if it does not interfere with theability of the peptide to elicit an immune response against thepolypeptide of the present invention. Examples of amino acid mirneticsinclude cyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantylalanine, adamantylacetic acid, and the like. See e.g. Morgan and Gainor,(1989) Ann. Repts. Med. Chem. 24:243-252, which is herein incorporatedby reference.

[0079] Individual amino acid residues may be incorporated in thepolypeptides of the present invention with peptide bonds or peptide bondmimetics. “Peptide bond mimetics” include peptide backbone modificationsof the amide nitrogen, the α-carbon, amide carbonyl, completereplacement of the amide bond, extensions, deletions or backbonecrosslinks. See e.g. Spatola (1983) CHEMISIRY AND BIOCHEMISTRY OF AMINOACIDS, PEPTIDES AND PROTEINS, Vol. VII, Weinstein ed., which is hereinincorporated by reference. The polypeptides of the present invention mayinclude an additional methionine as the first amino acid residue on theprotein amino terminus. The polypeptides may be truncated or containco-translational or post-translational surface modifications, such asthe addition of covalently attached sugars or lipids.

[0080] In preferred embodiments, the polypeptides of the presentinvention have a substantial sequence identity to the amino acidsequence set forth in SEQ ID NO:2 or SEQ ID NO:3. As used herein“sequence identity” means that two sequences are identical over a windowof comparison. The percentage of sequence identity is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical residuesoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

[0081] A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0082] The polypeptides of the present invention may comprise a signalor leader sequence at the N-terminal end of the polypeptide, whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification or identification of thepolypeptide such as a histidine tag, or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide may beconjugated to an immunoglobulin Fc region.

[0083] The polypeptides of the present invention and fragments thereofmay be made by conventional methods known in the art. The polypeptidesof the present invention may be manually or synthetically synthesizedusing conventional methods and devices known in the art. See e.g.,Stewart and Young (1984) SOLID PHASE PEPTIDE SYNTHESIS, 2 ed. Pierce,Rockford, Ill.; Merrifield, (1963) J. Am. Chem. Soc. 85:2149-2146, whichare herein incorporated by reference. Equipment for automated synthesisof polypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City,Calif.), and may beoperated according to the manufacturer's instructions. The compositionof the synthetic peptides may be confirmed by conventional methods inthe art, such as amino acid analysis or sequencing.

[0084] The polypeptides of the present invention may be purified fromnatural sources using conventional protein purification techniques suchas reverse phase high-performance liquid chromatography (HPLC),ion-exchange or immunoaffinity chromatography, filtration or sizeexclusion, or electrophoresis. See e.g., Scopes (1982) PROTEINPURIFICATION, Springer-Verlag, NY, which is herein incorporated byreference.

[0085] In some embodiments, the polypeptides of the present inventionmay be substantially purified. As used herein, a “substantiallypurified” compound refers to a compound that is removed from its nativeenvironment and is at least about 60% free, preferably about 75% free,and most preferably about 90% free from other macromolecular componentswith which the compound is naturally associated. A polypeptide of thepresent invention may be substantially purified by preparative highperformance liquid chromatography or other comparable techniquesavailable in the art. See e.g., Creighton, T. (1983) Proteins,Structures and Molecular Principles, VH Freeman and Co., New York, whichis herein incorporated by reference.

[0086] In other embodiments of the invention, polynucleotide sequencesor fragments thereof which encode RBP1L1 polypeptides, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of the polypeptide in appropriatehost cells. Due to the inherent degeneracy of the genetic code, otherDNA sequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

[0087] As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life that is longer than that of a transcript generated from thenaturally occurring sequence.

[0088] Sequences encoding a desired polypeptide may be synthesized, inwhole or in part, using conventional methods known in the art. SeeCaruthers, M. H., et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223;Horn, T., et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232; andRoberge, J. Y., et al. (1995) Science 269:202-204, which are hereinincorporated by reference. Commercially available automatedsynthesizers, such as ABI 431A Peptide Synthesizer (Perkin Elmer), maybe used. Moreover, the polynucleotide sequences of the present inventioncan be engineered using methods generally known in the art in order toalter polypeptide encoding sequences which modify the cloning,processing, or expression of the gene product, or a combination thereof.For example, DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. In addition, site-directed mutagenesis may beused to insert new restriction sites, alter glycosylation patterns,change codon preference, produce splice variants, introduce mutations,and the like.

[0089] In some embodiments, the polypeptide may be a fusion protein thatcomprises multiple polypeptides as described herein, or that comprisesat least one polypeptide as described herein and an unrelated sequence,such as a known tumor protein. Fusion proteins may generally be preparedusing standard techniques, including recombinant techniques and chemicalconjugation. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the polypeptide (an expression enhancer) athigher yields than the native recombinant protein. Certain preferredfusion partners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the polypeptide or to enable the polypeptide to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of thepolypeptide.

[0090] A peptide linker sequence may be employed to separate the firstand second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea, etal. (1985) Gene 40:39-46; Murphy, et al. (1986) PNAS USA 83:8258-8262;U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequencemay generally be from 1 to about 50 amino acids in length. Linkersequences are not required when the first and second polypeptides havenon-essential N-terminal amino acid regions that can be used to separatethe functional domains and prevent steric interference.

[0091] In order to express the polypeptides of the present invention,the nucleotide sequences encoding the polypeptide, or functionalequivalents, may be inserted into appropriate expression vector or acell, such as a breast cancer cell that naturally contains the RBP1L1polynucleotide sequence, may be induced to express the polypeptide bymethods known in the art.

[0092] A polynucleotide encoding a polypeptide of the present inventionis then inserted in to a vector such as a cloning vector or anexpression vector. An expression vector allows the polypeptide to beexpressed when present in a host. Either the expression vector or thehost may comprise the regulatory sequences necessary for expression ofthe polypeptide. Where the regulatory sequences are within theexpression vector, the regulatory sequences are operatively linked tothe sequence encoding the polypeptide. As used herein, “operably linked”means that the nucleotide sequence of interest is linked to at least oneregulatory sequence in a manner that allows the polypeptide to beexpressed in an in vitro transcription/translation system or in a hostcell. Regulatory sequences include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). See e.g.,Goeddel (1990) GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY,Academic Press, San Diego, Calif., which is herein incorporated byreference. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the desired expression levels of thepolypeptide, the compatibility of the host cell and the expressedpolypeptide, and the like.

[0093] The vectors can be designed for expressing the polypeptides ofthe present invention in prokaryotic or eukaryotic host cells such asbacterial cells, insect cells, plant cells, yeast cells, or mammaliancells. In preferred embodiments, the host cells are bacterial cells.Suitable host cells are discussed further in Goeddel supra; Baldari, etal. (1987) EMBO J. 6:229-234; Kurjan and Herskowitz (1982) Cell30:933-943; Schultz, et al. (1987) Gene 54:113-123; Smith, et al. (1983)Mol. Cell Biol. 3:2156-2165; Lucklow and Summers (1989) Virology170:31-39; Seed (1987) Nature 329:840; Kaufinan, et al. (1987) EMBO J.6:187-6195; Sambrook, et al. (2000) MOLECULAR CLONING: A LABORATORYMANUAL. Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., all of which are herein incorporated byreference.

[0094] Thus, the present invention also provides host cells comprisingpolynucleotides that encode the polypeptides of the present invention.Host cells include the progeny or potential progeny of the primary cellin which the polynucleotide was introduced. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope and meaningof host cell.

[0095] The present invention also provides antibodies and fragmentsspecific for the polypeptides, polynucleotides, and fragments thereof ofthe present invention and compositions comprising such. A polypeptide ofthe present invention may be used to prepare antibodies specific forcancerous cells and tumors by immunizing a suitable subject, e.g.,rabbit, goat, mouse or other mammal with the polypeptide by conventionalmethods known in the art. Large quantities of neutralizing antibodiescould be generated and then used in cancer immunotherapies by methodsknown in the art. The antibodies raised against the polypeptides of thepresent invention may be used to treat cancer by providing passiveimmunity or by creating immunotoxic compositions that are targeted tocancerous cells and tumors. Thus, the present invention providesantibodies that are raised against or derived from the polypeptides,polynucleotides, and fragments thereof of the present invention, andmethods of using thereof.

[0096] Antibodies of the present invention may be produced byconventional methods known in the art. See e.g., Coligan (1991) CURRENTPROTOCOLS IN IMMUNOLOGY. Wiley/Greene, NY; and Harlow and Lane (1989)ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press, NY; Stites,et al. (1986) BASIC AND CLINICAL IMMUNOLOGY. 4th ed. Lange MedicalPublications, Los Altos, Calif.; Goding (1986) MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE. 2d ed. Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256:495-497, which are hereinincorporated by reference. Therapeutic antibodies may be producedspecifically for clinical use in humans by conventional methods known inthe art. See Chadd, H. E. and S. M. Chamow (2001) Curr. Opin.Biotechnol. 12:188-194 and references therein, all of which are hereinincorporated by reference.

[0097] As used herein, “antibody” refers to immunoglobulin molecules andimmunologically active fragments that comprise an antigen binding sitewhich specifically binds an antigen, such as the epitope of the presentinvention, IKPSLGSKK (SEQ ID NO:3). Examples of immunologically activefragments of immunoglobulin molecules include F(ab) and F(ab′)₂fragments which may be generated by treating the antibody with an enzymesuch as pepsin. Polyclonal and monoclonal antibodies against thepolypeptides of the present invention may be made by conventionalmethods known in the art.

[0098] The present invention also provides binding agents, which includeantibodies and antigen-binding fragments thereof, that specifically bindto the polypeptide of the present invention. As used herein, anantibody, or antigen-binding fragment thereof “specifically binds” thepolypeptides of the present invention if the reaction is detectable withconvention assay methods known in the art and reactions, if any, withunrelated proteins under similar conditions are not detectable. As usedherein, “binding” refers to a noncovalent association between twoseparate molecules such that a complex is formed. The ability to bindmay be evaluated by, for example, determining a binding constant for theformation of the complex using methods known in the art.

[0099] Binding agents may be further capable of differentiating betweenindividuals with and without a cancer, such as breast, lung, colon,pancreas, and ovarian cancer, using the assays provided herein and knownin the art. Binding agents that indicate that an individual has cancerwill bind to a target antigen to generate a signal that is greater thana signal, if any, of a control. To determine whether a binding agentsatisfies this requirement, biological samples from patients with andwithout a cancer, as determined using standard clinical tests, may beassayed. Binding agents may be used alone or in combination to improvesensitivity or selectivity.

[0100] In preferred embodiments, the binding agent is an antibody or anantigen-binding fragment thereof that is specific for the RBP1L1polypeptide of the present invention and fragments thereof. Antibodiesand fragments thereof may be prepared by any of a variety of techniquesknown to those of ordinary skill in the art. See e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler and Milstein(1976) Eur. J. Immunol. 6:511-519, and improvements thereto, which areherein incorporated by reference.

[0101] Antibodies of the present invention may be coupled to one or moretherapeutic agents, such as drugs, differentiation inducers, toxins, andthe like. Suitable drugs include methotrexate, and pyrimidine and purineanalogs. Suitable differentiation inducers include phorbol esters andbutyric acid. Suitable toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein. The therapeutic agent may be coupled (e.g.,covalently bonded) to the antibody either directly or indirectly. Insome embodiments multiple molecules of a therapeutic agent are coupledto one antibody molecule. In some embodiments, more than one type ofagent may be coupled to one antibody.

[0102] A linker group may be used to couple the therapeutic agent andthe antibody. A linker group can function as a spacer to distance anantibody from an agent in order to avoid interference with bindingcapabilities. A linker group can also serve to increase the chemicalreactivity of a substituent on an agent or an antibody, and thusincrease the coupling efficiency. A variety of bifunctional orpolyfunctional reagents, both homo-and hetero-functional (such as thosedescribed in the catalog of the Pierce Chemical Co., Rockford, Ill.),may be employed as the linker group. Coupling may be effected, forexample, through amino groups, carboxyl groups, sulfhydryl groups oroxidized carbohydrate residues using conventional methods known in theart. Where a therapeutic agent is more potent when free from theantibody portion of the immunoconjugates of the present invention, itmay be desirable to use a linker group that is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups are known in the art. Carriers may be used to couple the bindingagent to the therapeutic agent or encapsulate the agents together.Suitable carriers include proteins such as albumins, peptides andpolysaccharides such as aminodextran, and the like.

[0103] The compositions of the present invention also include T-cellsspecific for the RPB1L1 polypeptide of the present invention. TheT-cells may be prepared by conventional methods known in the art. TheT-cells maybe stimulated with the RBP1L1 polypeptide, RBP1L1polynucleotide, an antigen presenting cell (APC) that expresses theRBP1L1 polypeptide, or a combination hereof. APCs may be transfected exvivo or in vivo with a polynucleotide of the present invention such thatthe polypeptide, or an immunogenic portion thereof, is expressed on thecell surface by conventional methods known in the art. See e.g. WO97/24447; and Mahvi, et al. (1997) Immunol. Cell Biol. 75:456-460, whichis herein incorporated by reference. Stimulation is performed underconditions and for a time sufficient to permit the generation of T-cellsthat are specific for the RBP1L1 polypeptide. T cells are considered tobe specific for the polypeptides of the present invention if the T cellsspecifically proliferate, secrete cytokines or kill target cells coatedwith the polypeptide or expressing a gene encoding the polypeptide. Tcell specificity may be evaluated using any of a variety of conventionaltechniques.

[0104] The polypeptides, polynucleotides, antibodies, or fragmentsthereof of the present invention may be used as an active agent inpharmaceutical compositions used to treat, prevent, inhibit, or modulatecancer. Preferred pharmaceutical compositions are those comprising atleast one polypeptide, polynucleotide, antibody, or fragment thereof ofthe present invention in a therapeutically effective amount, and apharmaceutically acceptable vehicle. Supplementary active agents canalso be incorporated into the compositions. Suitable supplementaryactive agents include pacitaxel, coumarin compounds, other tumorantigens such as HOM-MEL-40, NY-ESO-1, MAGE-1, GAGE, BAGE, antibodiesagainst other tumor antigens, and the like. The pharmaceuticalcompositions of the present invention may also include an adjuvant. Asused herein, an “adjuvant” refers to any substance which, whenadministered with or before the polypeptide, polynucleotide, or antibodyof the present invention, aids the polypeptide, polynucleotide, orantibody in its mechanism of action.

[0105] As used herein, “vehicle” and “carrier” are used interchangablyto indicate any and all solvents, dispersion media, vehicles, coatings,diluents, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, buffers, carrier solutions, suspensions, colloids, andthe like. As used herein, “pharmaceutically acceptable” vehicle orcarrier is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticalactive substances is well known in the art. See e.g. REMINGTON: THESCIENCE AND PRACTICE OF PHARMACY. 20^(th) ed. (2000) Lippincott Williams& Wilkins. Baltimore, Md., which is herein incorporated by reference.Except insofar as any conventional media or agent is incompatible withthe active ingredient, its use in the therapeutic compositions iscontemplated. Pharmaceutical carriers are preferably biocompatible, andmay also be biodegradable.

[0106] The compositions described herein may be administered as part ofa sustained release formulation that provides a slow release of theactive agent following administration. Time-delay or time-releasematerial is known in the art and includes glyceryl monostearate orglyceryl distearate alone or with a wax, ethylcellulose,hydroxypropylmethylcellulose, methylmethacrylate, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Likewise, the sustained release formulations may beprepared by conventional methods in the art. See e.g. Coombes et al.(1996) Vaccine 14:1429-1438, which is herein incorporated by reference.The sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix, containedwithin a reservoir surrounded by a rate controlling membrane, or acombination thereof. Other delayed-release vehicles includesupramolecular biovectors, which comprise a non-liquid hydrophilic coreand, optionally, an external layer comprising an amphiphilic compound,such as a phospholipid. The amount of active agent contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

[0107] The pharmaceutical compositions of the present invention mayinclude pharmaceutically acceptable salts, multimeric forms, prodrugs,active metabolites, precursors and salts of such metabolites of thepolypeptides, polynucleotides, antibodies, or fragments thereofdescribed herein.

[0108] The term “pharmaceutically acceptable salts” refers to salt formsthat are pharmacologically acceptable and substantially non-toxic to thesubject being treated with the compound of the invention.Pharmaceutically acceptable salts include conventional acid-additionsalts or base-addition salts formed from suitable non-toxic organic orinorganic acids and bases. Exemplary acid-addition salts include thosederived from inorganic acids such as hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid,and nitric acid, and those derived from organic acids such asp-toluenesulfonic acid, methanesulfonic acid, ethane-disulfonic acid,isethionic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid,succinic acid, citric acid, benzoic acid, 2-acetoxybenzoic acid, aceticacid, phenylacetic acid, propionic acid, glycolic acid, stearic acid,lactic acid, malic acid, tartaric acid, ascorbic acid, maleic acid,hydroxymaleic acids glutamic acid, salicylic acid, sulfanilic acid, andfumaric acid. Exemplary base-addition salts include those derived fromammonium hydroxides (e.g., a quaternary ammonium hydroxide such astetramethylammonium hydroxide), those derived from inorganic bases suchas alkali or alkaline earth-metal (e.g., sodium, potassium, lithium,calcium, or magnesium) hydroxides, and those derived from non-toxicorganic bases such as basic amino acids.

[0109] The term “multimer” refers to multivalent or multimeric forms ofactive forms of the compounds of the invention. Such “multimers” may bemade by linking or placing multiple copies of an active compound inclose proximity to each other, e.g., using a scaffolding provided by acarrier moiety. Multimers of various dimensions (i.e., bearing varyingnumbers of copies of an active compound) may be tested to arrive at amultimer of optimum size with respect to receptor binding. Provision ofsuch multivalent forms of active receptor-binding compounds with optimalspacing between the receptor-binding moieties may enhance receptorbinding. See e.g., Lee et al., (1984) Biochem. 23:4255, which is hereinincorporated by reference. The artisan may control the multivalency andspacing by selection of a suitable carrier moiety or linker units.Useful moieties include molecular supports comprising a multiplicity offunctional groups that can be reacted with functional groups associatedwith the active compounds of the invention. A variety of carriermoieties may be used to build highly active multimers, includingproteins such as BSA (bovine serum albumin) or HSA, peptides such aspentapeptides, decapeptides, pentadecapeptides, and the like, as well asnon-biological compounds selected for their beneficial effects onabsorbability, transport, and persistence within the target organism.Functional groups on the carrier moiety, such as amino, sulfhydryl,hydroxyl, and alkylamino groups, may be selected to obtain stablelinkages to the compounds of the invention, optimal spacing between theimmobilized compounds, and optimal biological properties.

[0110] “A pharmaceutically acceptable prodrug” is a compound that may beconverted under physiological conditions or by solvolysis to thespecified compound or to a pharmaceutically acceptable salt of suchcompound. “A pharmaceutically active metabolite” is intended to mean apharmacologically active product produced through metabolism of aspecified compound or salt thereof in the body. Prodrugs and activemetabolites of a compound may be identified using routine techniquesknown in the art. See e.g., Bertolini, G. et al., (1997) J. Med. Chem.40:2011-2016; Shan, D. et al., J. Pharm. Sci., 86(7):765-767; BagshaweK., (1995) Drug Dev. Res. 34:220-230; Bodor, N., (1984) Advances in DrugRes. 13:224-331; Bundgaard, H., Design of Prodrugs (Elsevier Press,1985); and Larsen, I. K., Design and Application of Prodrugs, DrugDesign and Development (Krogsgaard-Larsen et al., eds., Harwood AcademicPublishers, 1991), which are herein incorporated by reference.

[0111] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Suitable pharmaceuticalformulations for particular routes of administration are well known inthe art. See e.g. REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY.20^(th) ed. (2000) Lippincott Williams & Wilkins. Baltimore, Md., whichis herein incorporated by reference.

[0112] The compositions of the invention may be manufactured in mannersgenerally known for preparing pharmaceutical compositions, e.g., usingconventional techniques such as mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing. Pharmaceutical compositions may be formulated in aconventional manner using one or more physiologically acceptablecarriers, which may be selected from excipients and auxiliaries thatfacilitate processing of the active agents.

[0113] The pharmaceutical compositions of the present invention may beprovided in dosage unit forms in unit-dose or multi-dose containers,such as sealed ampoules or vials. Such containers are preferablyhermetically sealed to preserve sterility of the formulation until use.In general, the compositions of the present invention may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, the compositions may be stored in a freeze-driedcondition requiring only the addition of a sterile liquid carrier priorto use. As used herein, “dosage unit form” refers to physically discreteunits suited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

[0114] It will be appreciated that the actual dosages of thepolynucleotides, polypeptides, antibodies, and fragments thereof used inthe compositions of this invention will vary according to the particularcomplex being used, the particular composition formulated, the mode ofadministration, and the particular site, host, and disease beingtreated. Optimal dosages for a given set of conditions may beascertained by those skilled in the art using conventionaldosage-determination tests in view of experimental data. Administrationof prodrugs may be dosed at weight levels that are chemically equivalentto the weight levels of the fully active forms.

[0115] The biological activity of the polynucleotides, polypeptides,antibodies, fragments thereof, and compositions of the present inventionmay be measured by any of the methods available to those skilled in theart, including in vitro and in vivo assays and those provided herein.Other pharmacological methods may also be used to determine the efficacyof the polynucleotides, polypeptides, antibodies, fragments thereof, andcompositions of the present invention as anti-neoplastic agents andcancer immunotherapeutics.

[0116] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals. For example, the LD₅₀ (the dose lethal to 50% ofthe population), and the ED₅₀ (the dose therapeutically effective in 50%of the population) may be determined by conventional methods in the art.The dose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds thatexhibit large therapeutic indices are preferred. While compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissue in order to minimize potential damage to nonnal, non-cancerous,or benign cells and, thereby, reduce side effects.

[0117] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0118] The pharmaceutical compositions of the present invention includeimmunogenic compositions. The immunogenic compositions include an activeimmunizing agent, such as a polypeptide of the present invention, or apassive immunizing agent, such as an antibody raised against orspecifically binds the polypeptide of the present invention. Theimmunogenic composition may elicit an immune response that need not beprotective or the immunogenic composition may provide passive immunity.A vaccine elicits a local or systemic immune response that is protectiveagainst subsequent challenge by the immunizing agent such as thepolypeptides of the present invention, or an immunologicallycross-reactive agent. Accordingly, as used herein, an “immunogeniccomposition” can refer to vaccines as well as antibodies. A protectiveimmune response maybe complete or partial, i.e. a reduction in symptomsas compared with an unvaccinated mammal. Conventional methods in the artmay be used to determine the feasibility of using the polypeptides ofthe present invention as a cancer or tumor vaccine.

[0119] Thus, the present invention provides immunogenic compositionscomprising the polypeptides, polynucleotides, antibodies, or fragmentsthereof of the present invention that may be used to treat, prevent,inhibit, or modulate cancer. As used herein, an “immunogenic amount” isan amount that is sufficient to elicit an immune response in a subjectand depends on a variety of factors such as the immunogenicity of thepolypeptide, the manner of administration, the general state of healthof the subject, and the like. The typical immunogenic amounts forinitial and boosting immunization for therapeutic or prophylacticadministration ranges from about 0.01 mg to about 0.1 mg per about 65-70kg body weight of a subject. Examples of suitable immunization protocolsinclude initial immunization injections at time 0 and 4 or initialimmunization injections at 0, 4, and 8 weeks, which initial immunizationinjections may be followed by further booster injections at 1 or 2years.

[0120] A variety of immunostimulants maybe employed in the immunogeniccompositions of the present invention. For example, an immunostimulantmay be included. An “immunostimulant” is any substance that enhances orpotentiates an immune response (antibody and/or cell-mediated) to anexogenous antigen. Examples of immunostimulants include adjuvants,biodegradable microspheres (e.g., polylactic galactide), liposomes,cytokines, interleukins, and chemokines. Most adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a stimulator of immune responses,such as lipid A, Bortadella pertussis or Mycobacterium tuberculosisderived proteins. Suitable adjuvants include Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.);Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKlineGlaxo); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A; and cytokines, such asGM-CSF or interleukin-2,-7, or -12, may be used as adjuvants. Othersuitable adjuvants include Montanide ISA 720 (Seppic, France), SAF(Chiron, Emeryville, Calif.), ISCOMS (CSL), MF-59 (Chiron), the SBASseries of adjuvants, such as SBAS-2 or SBAS-4 (SmithKlineGlaxo), Detox,RC-529, other aminoalkyl glucosaminide 4-phosphates (AGPs); incompleteN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP),N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipa-Imitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, MTP-PE); and RIBI, which comprise three componentsextracted from bacteria, monophosphoryl lipid A, trehalose dimycolateand cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.The effectiveness of an adjuvant may be determined by conventionalmethods in the art.

[0121] The vaccines of the present invention maybe prepared byconventional methods known in the art. See e.g. M. F. Powell and M. J.Newman, eds. VACCINE DESIGN Plenum Press (NY, 1995), which is hereinincorporated by reference. The immunogenic compositions of the presentinvention may further comprise other agents, which may be biologicallyactive or inactive. For example, one or more immunogenic portions ofother tumor antigens, either incorporated into a fusion polypeptide oras a separate compound, may be included in the compositions.

[0122] A variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets tumor cells. Suitabledelivery vehicles include antigen presenting cells (APCs), such asdendritic cells, macrophages, B cells, monocytes and other cells thatmay be engineered to be efficient APCs. Such cells may be geneticallymodified to increase the capacity for presenting the antigen, to improveactivation of the T cell response, maintenance of the T cell response,to have anti-tumor effects per se, to be immunologically compatible withthe receiver, or a combination thereof. APCs may generally be isolatedfrom any of a variety of biological fluids and organs, including tumorand peritumoral tissues, and may be autologous, allogeneic, syngeneic,or xenogeneic cells by methods known in the art.

[0123] The present invention also provides polypeptides,polynucleotides, antibodies, or compositions of the present inventionthat may be provided in kits along with instructions for use. A kitcomprising a pharmaceutical composition may include the pharmaceuticalcomposition as a single dose or multiple doses. The kit may include adevice for delivering the pharmaceutical composition. The device may bea multi-chambered syringe for intramuscular delivery, a microneedle orset of microneedle arrays for transdermal delivery, a small balloon forintranasal delivery, or a small aerosol generating device for deliveryby inhalation.

[0124] The polynucleotides, polypeptides, antibodies, and compositionsof the present invention may be used as active agents in therapeuticmethods to treat cancer in a subject. Generally, these therapeuticmethods comprise administering to the subject a therapeuticallyeffective amount of at least one active agent.

[0125] As used herein, a “therapeutically effective amount” refers to anamount of an active agent that may be used to treat cancer in a subjectas compared to a control. As used herein, “treating cancer” includesinhibiting, preventing, modulating, or reducing the growth or spread ofcancer as well as reducing or maintaining the amount of cancerous cellsin a subject or biological sample as compared to a control. Again, theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including the severityand stage of the cancer, previous treatments, the general health and ageof the subject, and the like. A therapeutically effective amount may bereadily determined by conventional methods known in the art. It shouldbe noted that treatment of a subject with a therapeutically effectiveamount of a polypeptide, a polynucleotide, or an antibody of the presentinvention can include a single treatment or, preferably, can include aseries of treatments.

[0126] The polynucleotides, polypeptides, antibodies, and compositionsof the present invention may be used in cancer immunotherapies. Inpreferred embodiments, the cancer is breast, lung, colon, pancreas, orovarian cancer. In particular, the polynucleotides, polypeptides,antibodies, and compositions of the present invention may beadministered to a subject, preferably human. The subject may or may notbe afflicted with cancer. Accordingly, the polynucleotides,polypeptides, antibodies, and compositions of the present invention maybe used to prevent the development of a cancer in a subject or to treata subject afflicted with a cancer. A cancer may be diagnosed usingcriteria generally accepted in the art, including the presence of amalignant tumor, or the methods described herein. The polynucleotides,polypeptides, antibodies, and compositions of the present invention maybe administered prior to, during, or after surgical removal of primarytumors or treatment such as administration of radiotherapy orconventional chemotherapeutic drugs, or a combination thereof.Administration may be by any suitable method, including administrationby intravenous, intraperitoneal, intramuscular, subcutaneous,intranasal, intradermal, anal, vaginal, topical and oral routes.

[0127] The polynucleotides, polypeptides, antibodies, and compositionsof the present invention may be used in combination with or as asubstitution for conventional cancer therapies. For example, thepolynucleotides, polypeptides, antibodies, and compositions of thepresent invention may also be used alone or in combination with asupplementary active compound such as methotrexate, taxol, and the like,to treat breast, lung, colon, pancreas, or ovarian cancer in a subject.Likewise, the polynucleotides, polypeptides, antibodies, andcompositions of the present invention may be used in combination with orin place of surgical procedures.

[0128] Cancer immunotherapies of the present invention may be broadlyclassified as adoptive, passive, and active. The therapeutic methods ofthe present invention may be active immunotherapy, in which treatmentrelies on the in vivo stimulation of the endogenous host immune systemto react against tumors with the administration of immuneresponse-modifying agents, such as the polypeptides and polynucleotidesof the present invention. Active immunotherapies of the presentinvention include administering the antigenic epitope of the presentinvention, a polynucleotide encoding the antigenic epitope, adjuvants,immunostimulants, and the like. In some embodiments, other tumorassociated antigens, such as HOM-MEL-40, NY-ESO-1, MAGE-1, GAGE, BAGE,KASIFLK (SEQ ID NO:5), GLQKASIFLK (SEQ ID NO:6), KASIFLKTRV (SEQ IDNO:7), and the like, may be administered along with the IKPSLGSKK (SEQID NO:3) or variants thereof. In some preferred embodiments, the IgMantibody response in the subject being treated is activated or enhanced.

[0129] Alternatively, the therapeutic methods of the present inventionmay be passive immunotherapy, in which treatment involves the deliveryof agents with established tumor-immune reactivity (such as effectorcells or antibodies) that can directly or indirectly mediate antitumoreffects and does not necessarily depend on an intact host immune system.The T cell receptors and antibody receptors specific for thepolypeptides of the present invention may be cloned, expressed, andtransferred into other vectors or effector cells for adoptiveimmunotherapy. The polypeptides provided herein may also be used togenerate antibodies or anti-idiotypic antibodies for passiveimmunotherapy by methods known in the art. Passive immunotherapies ofthe present invention include administering antibodies against IKPSLGSKK(SEQ ID NO:3) or a variant thereof alone or coupled to toxins,chemotherapeutic agents, radioactive isotopes, and the like. In someembodiments, antibodies against other tumor associated antigens, such asHOM-MEL-40, NY-ESO-1, MAGE-1, GAGE, BAGE, KASIFLK (SEQ ID NO:5),GLQKASIFLK (SEQ ID NO:6), KASIFLKTRV (SEQ ID NO:7), and the like, may beadministered along with the antibodies against IKPSLGSKK (SEQ ID NO:3)or variants thereof. In some embodiments, lymphokines and other types ofimmunostimulants may be administered. See e.g., Bajorin et al. (1988)Proc. Annu. Meet. Am. Soc. Clin. Oncol. 7: A967.

[0130] Adoptive immunotherapies of the present invention includeisolating a subject's circulating lymphocytes or tumor infiltratedlymphocytes and activating the lymphocytes by conventional methods knownin the art and then administering the activated lymphocytes to a subjectto be treated.

[0131] In some embodiments, the present invention provides methods ofenhancing the immune response in a subject comprising the steps ofcontacting at least one lymphocyte with a polypeptide comprisingIKPSLGSKK (SEQ ID NO:3) or a variant thereof. In some embodiments, otherantigenic polypeptides, such as HOM-MEL-40, NY-ESO-1, MAGE-1, GAGE,BAGE, KASIFLK (SEQ ID NO:5), GLQKASIFLK (SEQ ID NO:6), KASIFLKTRV (SEQID NO:7), and the like, may be contacted with the lymphocyte along withthe a polypeptide comprising IKPSLGSKK (SEQ ID NO:3) or a variantthereof. Additional compounds, such as immunostimulants may beadministered to the subject.

[0132] Routes and frequency of administration of the therapeuticcompositions described herein, as well as dosage, will vary fromindividual to individual, and may be readily established using standardtechniques. In general, the pharmaceutical compositions and vaccines maybe administered by injection, intranasally, or orally. Preferably,between 1 and 10 doses may be administered over a 52-week period.Preferably, 6 doses are administered, at intervals of 1 month, andbooster vaccinations may be given periodically thereafter. Alternateprotocols may be appropriate for individual patients.

[0133] A suitable dose is an amount of a polynucleotide, polypeptide,antibody, or composition of the present invention that, whenadministered as described above, is capable of promoting an anti-tumorimmune response, and is at least about 10% or more above the basallevel. In general, for pharmaceutical compositions and vaccinescomprising one or more polypeptides, the amount of each polypeptidepresent in a dose ranges from about 25 μg to about 5 mg per kg of thesubject. Suitable dose sizes will vary with the size of the subject, butwill typically range from about 0.1 ml to about 5 ml. Methods of usingthe vaccines of the present invention are also contemplated. Suitablevaccines are capable of causing an immune response that leads to animproved clinical outcome, such as more frequent remissions, complete orpartial or longer disease-free survival, in vaccinated patients ascompared to non-vaccinated patients.

[0134] Responses to the thereapeutic methods of the present inventioncan be monitored by measuring the anti-tumor antibodies in a patient orby vaccine-dependent generation of cytolytic effector cells capable ofkilling the patient's tumor cells in vitro. Immune responses maygenerally be evaluated using standard proliferation, cytotoxicity orcytokine assays, which may be performed using samples obtained from asubject before and after treatment. Other methods known in the art formonitoring the effect of a given therapeutic method are alsocontemplated.

[0135] As used herein, a “therapeutically effective amount” of apolynucleotide, polypeptide, antibody, or composition of the presentinvention is an amount which treats cancer in a subject as compared to acontrol. As defined herein, a therapeutically effective amount of acompound of the present invention may be readily determined by one ofordinary skill by routine methods known in the art. For example, atherapeutically effective amount of a compound of the invention rangesfrom about 0.1 to about 1000 mg/kg body weight, such as, about 1 toabout 750 mg/kg body weight, about 1 to about 500 mg/kg body weight,about 1 to about 350 mg/kg body weight, about 1 to about 200 mg/kg bodyweight, about 1 to about 100 mg/kg body weight, about 1 to about 50mg/kg body weight, about 1 to about 10 mg/kg body weight, about 1 toabout 5 mg/kg body weight, and the like. The skilled artisan willappreciate that certain factors may influence the dosage required toeffectively treat a subject, including but not limited to the severityof the disease or disorder, previous treatments, the general healthand/or age of the subject, and other diseases present.

[0136] Preferred topical concentrations include about 0.1% to about 10%in a formulated salve. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present.

[0137] The therapeutic methods of the present invention may include asingle treatment or a series of treatments. For example, a subject maybe treated with an immunogenic composition of the invention at leastonce. However, the subject maybe treated with the immunogeniccomposition from about one time per week to about once daily for a giventreatment period. The length of the treatment period will depend on avariety of factors such as the stage and aggressiveness of the cancer.It will also be appreciated that the effective dosage of the compoundused for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentby standard diagnostic assays known in the art. In some instanceschronic administration may be required.

[0138] The polynucleotides, polypeptides, antibodies, and fragmentsthereof of the present invention may be used to detect, diagnose, ormonitor cancer in a subject. Specifically, a cancer may be detected ormonitored in a subject based on the presence or amount of one or morepolynucleotides, polypeptides, or fragments thereof of the presentinvention in a biological sample obtained from the patient. Thus, thepolynucleotides, polypeptides, antibodies, and fragments thereof of thepresent invention may be used as markers to indicate the presence orabsence of a cancer, such as breast, lung, colon, pancreas, and ovariancancer, as well as other cancers. The binding agents provided hereingenerally permit detection of the level of antigen that binds to theagent in the biological sample. Polynucleotide primers and probes may beused to detect the differential expression of mRNA encoding a tumorprotein using assays known in the art, The differential expression ofthe epitope of the present invention is indicative of the presence orabsence of a cancer. Generally, the presence or absence of a cancer in asubject may be determined by contacting a biological sample obtainedfrom a subject with a binding agent and determining whether theantigenic epitope of the present invention is differentially expressedin the biological sample as compared to a control. As sample wherein theantigenic epitope is differentially expressed is indicative of cancer.

[0139] In some embodiments, the binding agent used in the assays of thepresent invention is immobilized on a solid support to bind to andremove the polypeptide from the remainder of a sample. The boundpolypeptide may then be detected using a detection reagent that containsa reporter group and specifically binds to the binding agent/polypeptidecomplex. Such detection reagents may comprise, for example, a bindingagent that specifically binds to the polypeptide or an antibody or otheragent that specifically binds to the binding agent, such as ananti-immunoglobulin, protein G, protein A, or a lectin. Alternatively, acompetitive assay may be utilized, in which a polypeptide is labeledwith a reporter group and allowed to bind to the immobilized bindingagent after incubation of the binding agent with the sample. The extentto which components of the sample inhibit the binding of the labeledpolypeptide to the binding agent is indicative of the reactivity of thesample with the immobilized binding agent. Suitable polypeptides for usein assays include full-length RBP1L1 polypeptide and fragments thereofto which the binding agent binds.

[0140] The solid support may be any material known to those of ordinaryskill in the art to which a polypeptide may be attached. For example,the solid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead, fiber, or disc, such as glass, fiberglass, latex, orplastic such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor. The binding agent may beimmobilized on the solid support by conventional methods known in theart. As used herein, “immobilization” refers to both noncovalentassociation, such as adsorption, and covalent attachment (which may be adirect linkage between the agent and functional groups on the support ormay be a linkage by way of a cross-linking agent). Adsorption may beachieved by contacting the binding agent, in a suitable buffer, with thesolid support for a suitable amount of time sufficient to immobilize anadequate amount of binding agent. Covalent attachment of binding agentto a solid support may be achieved by first reacting the support with abifunctional reagent that will react with both the support and afunctional group, such as a hydroxyl or amino group, on the bindingagent. See e.g. PIERCE IMMUNOTECHNOLOGY CATALOG AND HANDBOOK (1991) atA12-A13, which is herein incorporated by reference.

[0141] The presence of T cells that specifically react with RBP1L1polypeptides in a biological sample may indicate a cancer in a subject.A cancer may also be detected based on the level of mRNA encoding RBP1L1in a biological sample. Techniques for both PCR based assays andhybridization assays are well known in the art. See e.g. Mullis et al.(1987) Cold Spring Harbor Symp. Quant. Biol., 51:263; and Erlich ed.,PCR TECHNOLOGY, Stockton Press, NY, 1989, which are herein incorporatedby reference.

[0142] The compositions of the present invention may be used as markersfor the progression of cancer. In this embodiment, the assays asdescribed above. for the diagnosis of a cancer may be performed overtime, and the change in the level of at least one reactive polypeptideor polynucleotide is evaluated. For example, the assays may be performedevery 24 to 72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosesubjects in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

[0143] Certain in vivo diagnostic assays may be performed directly on atumor. One such assay involves contacting tumor cells with a bindingagent. The bound binding agent may then be detected directly orindirectly via a reporter group. Such binding agents may also be used inhistological applications. Alternatively, polynucleotide probes may beused within such applications.

[0144] As noted above, to improve sensitivity, multiple tumor proteinmarkers may be assayed within a given sample. It will be apparent thatbinding agents specific for different proteins provided herein may becombined within a single assay. Further, multiple primers or probes maybe used concurrently. The selection of tumor protein markers may bebased on routine experiments to determine combinations that results inoptimal sensitivity. In addition, or alternatively, assays for tumorproteins provided herein may be combined with assays for other knowntumor antigens.

[0145] The present invention further provides kits for use within any ofthe above diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to an ovarian tumor protein. Suchantibodies or fragments may be provided attached to a support material,as described above. One or more additional containers may encloseelements, such as reagents or buffers, to be used in the assay. Suchkits may also, or alternatively, contain a detection reagent asdescribed above that contains a reporter group suitable for direct orindirect detection of antibody binding.

[0146] Alternatively, a kit may be designed to detect the level of mRNAencoding RBP1L1 in a biological sample. Such kits generally comprise atleast one oligonucleotide probe or primer that hybridizes to apolynucleotide encoding RBP1L1. Such an oligonucleotide may be used, forexample, within a PCR or hybridization assay. Additional components thatmay be present within such kits include a second oligonucleotide and/ora diagnostic reagent or container to facilitate the detection of apolynucleotide encoding RBP1L1.

[0147] In some embodiments, antibodies that specifically bind RBP1L1 maybe used for the diagnosis of cancer, or in assays to monitor patientsbeing treated with RBP1L1, agonists, antagonists or inhibitors. Theantibodies useful for diagnostic purposes may be prepared in the samemanner as those described above for therapeutics. Diagnostic assays forRBP1L1 include methods that utilize the antibody and a label to detectRBP1L1 in human body fluids or extracts of cells or tissues. Theantibodies may be used with or without modification, and may be labeledby joining them, either covalently or non-covalently, with a reportermolecule. A wide variety of reporter molecules that are known in the artmay be used.

[0148] A variety of protocols including ELISA, RIA, and FACS formeasuring RBP1L1 are known in the art and provide a basis for diagnosingaltered or abnormal levels of RBP1L1 expression. Normal or standardvalues for RBP1L1 expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with.antibody to RBP1L1 under conditions suitable for complex formation.The amount of standard complex formation may be quantified by variousmethods known in the art, but preferably by photometric methods.Quantities of RBP1L1 expressed in subject, control and disease samplesfrom biopsied tissues are compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease.

[0149] In another embodiment of the invention, the polynucleotidesencoding RBP1L1 may be used for diagnostic purposes. The polynucleotidesthat may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofRBP1L1 may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofRBP1L1, and to monitor regulation of RBP1L1 levels during therapeuticintervention.

[0150] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding RBP1L1 or closely related molecules, may be used to identifynucleic acid sequences that encode RBP1L1. The specificity of the probewill determine whether the probe identifies only naturally occurringsequences encoding RBP1L1, alleles, or related sequences.

[0151] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the RBP1L1 encoding sequences. The hybridization probes of thesubject invention may be DNA or RNA and derived from the polynucleotidesequence of SEQ ID NO:1 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring RBP1L1.

[0152] Means for producing specific hybridization probes for DNAsencoding RBP1L1 include the cloning of nucleic acid sequences encodingRBP1L1 or RBP1L1 derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radiolabels such as ³²p or ³⁵S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0153] Polynucleotide sequences encoding RBP1L1 may be used for thediagnosis of cancer, such as breast, lung, colon, pancreas, and ovariancancer. The polynucleotide sequences encoding RBP1L1 may be used inSouthern or Northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered RBP1L1 expression. Such qualitative or quantitative methods arewell known in the art.

[0154] In order to provide a basis for the diagnosis of cancerassociated with expression of RBP1L1, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes RBP1L1,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0155] Once cancer is diagnosed and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

[0156] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actual toclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0157] Additional diagnostic uses for oligonucleotides designed from thesequences encoding RBP1L1 may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′ 3′) and another with antisense(3′ 5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

[0158] Methods that may also be used to quantitate the expression ofRBP1L1 include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated. See Melby, P. C., etal. (1993) J. Immunol. Methods 159:235-244; and Duplaa, C., et al.(1993) Anal. Biochem. 229-236, which are herein incorporated byreference. The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0159] In another embodiment of the invention, the nucleic acidsequences that encode RBP1L1 may also be used to generate hybridizationprobes that are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques includingFISH, FACS, or artificial chromosome constructions, such as yeastartificial chromosomes, bacterial artificial chromosomes, bacterial P1constructions or single chromosome cDNA libraries. See Price, C. M.,(1993) Blood Rev. 7:127-134; and Trask, B. J., (1991) Trends Genet.7:149-154; Verma, et al. (1988) HUMAN CHROMOSOMES: A MANUAL OF BASICTECHNIQUES, Pergamon Press, New York, which are herein incorporated byreference. Correlation between the location of the gene encoding RBP1L1on a physical chromosomal map and a specific disease, or predispositionto a specific disease, may help delimit the region of DNA associatedwith that genetic disease. The nucleotide sequences of the subjectinvention may be used to detect differences in gene sequences betweennormal, carrier, or affected individuals.

[0160] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

[0161] In another embodiment of the invention, RBP1L1, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenRBP1L1 and the agent being tested, may be measured.

[0162] Another technique for drug screening that may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564 and by using cells described in WO 99/15650. Inanother embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding RBP1L1 specificallycompete with a test compound for binding RBP1L1.

[0163] A variety of protocols for detecting and measuring the expressionof polynucleotide-encoded products, using either polyclonal ormonoclonal antibodies specific for the product are known in the art. SeeHampton, R., et al. (1990) SEROLOGICAL METHODS, A LABORATORY MANUAL, APSPress, St Paul, Minn.; and Maddox, D. E., et al. (1983) J. Exp. Med.158:1211-1216, which are herein incorporated by reference.

[0164] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Methods for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits such as those available from Amersham Pharmacia Biotech, Promegaand US Biochemical Corp. Suitable reporter molecules or labels, whichmay be used, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0165] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1 Human IRG Purification and MCF-7 cDNA Library Immunoscreening

[0166] An IgG antibody fraction was prepared from serum of a patientwith breast cancer. See Cao, J., et al. (1999) Breast Cancer Res. Treat.53:279-290, which is herein incorporated by reference. Antibodiesagainst Escherichia coli proteins and the heptapeptide KASIFLK (SEQ IDNO:5) in the IgG fraction were, respectively, removed first with anaffinity column containing an immobilized bacterial lysate from E. coliY1090 and then with a KASIFLK peptide affinity column. A λgt11 cDNAexpression library from MCF-7 cells was screened with the resultingpurified IgG (at 20 μg/ml, about a 1:400 dilution of the original serum)by the method of Young and Davis. See Young and Davis (1983) PNAS USA80:1194-1198, which is herein incorporated by reference.

[0167] 4×10⁶ plaques of the λgt11 MCF-7 cDNA expression library werescreened with human IgG purified from the serum of a patient with breastcancer and two immunopositive clones, 131 and 151, were detected. PCRanalysis of both clones using λgt11 forward and reverse primers revealeda 4.0-kilobase (kb) insert. DNA from clones 131 and 151 was transfectedinto lysogenic E. coli Y1089 and expression of the β-galactosidasefusion protein induced with IPTG.

[0168] Western blot analysis of the whole cell lysates with the purifiedIgG detected a fusion protein of about 128 kDa for each clone. Giventhat β-galactosidase is 116 kDa, the size of the open reading frame ofeach clone would be about 12 kDa. Therefore, only a small portion of theinsert of about 4.0-kb appeared to encode the protein detected by thepurified IgG.

EXAMPLE 2 Expression and Analysis of Fusion Proteins

[0169]E. coli Y1090 and Y1089 were used for screening and proteinexpression of λgt11 recombinants, respectively. After immunopositiverecombinants clones 131 and 151 were detected and plaques purified, theλgt11 recombinants were infected in lysogenic bacterial strain Y 1089that enhances the frequency of phage lysogeny. Fusion proteins wereexpressed in E. coli Y1089 as described by Oka, T., et al. (1994) CancerRes. 54:3511-3515, which is herein incorporated by reference. Proteinsamples were separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) on 4%-20% gradient gels, and proteins werestained with Coomassie blue R-250 or electrotransferred tonitrocellulose membranes for Western blot analysis. The Western blotmethod of assessing IgG antibody reactivity against the β-galactosidasefusion-protein antigens has been described by Kikumoto, Y., et al.(1995) Hybridoma 14:45-50, which is herein incorporated by reference.Peroxidase-conjugated rabbit anti-human IgG (Pierce, Rockford, Ill.) wasused as the secondary antibody.

[0170]FIG. 1 provides the results of expression of immunopositive fusionproteins in E. coli. The lanes designated as “A” in FIG. 1 provide thesodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)followed by Coomasie brilliant blue staining. The lanes designated as“B” in FIG. 1 provide the Western blot analysis with the purified humanIgG. The lanes designated “a” are the lysate of isopropylβ-D-thiogalactopyranoside (IPTG)-induced lysogen from clone 131. Thelanes designated “b” are the lysate of IPTG-induced lysogen from clone151. The band at 128 kDa represents the fusion protein containing a 107amino acid insert. Molecular mass markers (in kDa) are indicated in theleft lane.

EXAMPLE 3 Sequence Analysis of Immunoreactive Clones

[0171] DNA inserts from immunoreactive λgt11 clones 131 and 151 wereamplified by polymerase chain reaction (PCR) with λgt11 forward andreverse primers and cloned into the PCRII plasmid using the TA cloningstrategy (Invitrogen Corp., Carlsbad, Calif.). Total RNA from MCF-7 andperipheral blood mononuclear cells (PBMCs) were amplified by reversetranscription RT-PCR, and cDNA from human ovarian cells (Marathon-Ready™kit, Clontech, Palo Alto, Calif.) was amplified by PCR. Products fromall reactions were cloned into the PCRII plasmid and sequencedcompletely. DNA and amino acid sequences were compared with sequences inGenBank using the BLAST tool at National Center for BiotechnologyInformation (NCBI). Publication output for RBP1 and RBP1L1 proteinsequences alignment was generated with the Baylor College of Medicinesearch launcher BOXSHADE program.

[0172] Northern blot analysis was performed on human multiple tissuenorthern (MTN II) and human endocrine system MTN blots (Clontech). Onthese blots, each lane contained about 2 μg of poly(A)+ RNA from humanspleen, thymus, prostate, testis, ovary, small intestine, colon,peripheral blood leukocyte, pancreas, adrenal medulla, thyroid, adrenalcortex, or stomach. Membranes were hybridized with a ³²P-labeled441-base-pair (bp) fragment of RBP1L1 cDNA that was amplified by PCRfrom the immunopositive clone 15 1. Blots were washed twice in 0.1%standard saline citrate (SSC) and 0.1 % SDS for 20 minutes and thenexposed to Kodak BIOMAX film at −80° C. with an intensifying screen for36 hours. As a loading control, blots were stripped and reprobed withthe control glyceraldehyde 3-phosphoate dehydrogenase (G3PDH) gene toprove RNA integrity.

[0173] A stop codon at nucleotide 322 in clone 151 was investigated todetermine whether it was due to a somatic mutation in the gene or causedby mispairing during cDNA synthesis by sequencing RT-PCR productsamplified from MCF-7 and PBMC RNA. Total RNA (1 μg) from MCF-7 cells andPBMCs was primed with (dT)₁₂₋₁₈ and reverse transcribed with SuperScriptRT (Gibco, Rockville, Md.). A 470-bp segment of RBP1L1, spanning theregion, which contained the stop codon in clone 151, was amplified byPCR with the following sequence-specific primers: sense =5′-ATGGAGGAGGAGAGGAATATAATACCAAG-3′ (SEQ ID NO:8) antisense =5′-CTGAAATGGTGGTTTGGACAAGCGCCGA-3′ (SEQ ID NO:9)

[0174] (Operon Technologies, Alameda, Calif.).

[0175] PCR was performed to denature it at 94° C., 2 minutes; followedby 35 amplification cycles of the denatured product at 94° C., 30seconds; annealing temperature at 68° C. for 30 seconds and extension at72° C., 1 minutes with a final extension at 72° C., 5 minutes in aPerkin-Elmer thermal cycler. Several RT-PCR products were sequenced todetermine whether they also encoded a stop codon at the same position.In order to identify the remaining 5′ coding sequence of RBP1L1 and the5′-untranslated region (UTR), rapid amplification of 5′ cDNA ends (5′RACE) was performed on cDNA from human ovarian cells (Marathon-Ready™,Clontech) using a 5′ primer that had been designed based on the sequenceof the rat retinablastoma binding protein-1-related gene (rRBP1-R),which shares 84% sequence identity with the partial RBP1L1 sequence.

[0176] PCR was performed to denature it at 94° C., 2 minutes; followedby 35 amplification cycles of the denatured product at 94° C., 30seconds; annealing temperature at 68° C. for 30 seconds and extension at72° C., 1 minutes with a final extension at 72° C., 5 minutes in aPerkin-Elmer thermal cycler. Several RT-PCR products were sequenced todetermine whether they also encoded a stop codon at the same position.In order to identify the remaining 5′ coding sequence of RBP1L1 and the5′-untranslated region (UTR), rapid amplification of 5′ cDNA ends (5′RACE) was performed on cDNA from human ovarian cells (Marathon-Ready™,Clontech) using a 5′ primer that had been designed based on the sequenceof the rat retinablastoma binding protein-1-related gene (rRBP1-R),which shares 84% sequence identity with the partial RBP1L1 sequence.

[0177] A sense primer: (SEQ ID NO:10)5′-AGAGTCACCATGAAGGCCCTTGATGATGAGC-3′

[0178] corresponding to the 5′ end of rRBP1-R (nucleotides 30-57) and anantisense primer: 5′-TGGGATTATATTCCTCTCCTCCTCCATC-3′ (SEQ ID NO:11)

[0179] corresponding to the 5′ end of the incomplete human RBP1L1sequence (nucleotides 12-39 of clone 151) were used. Sequencing of theresulting reaction product identified the remaining 5′ cDNA sequence ofhuman RBP1L1.

[0180] To obtain the 5′ UTR sequence, the region was amplified by usingan adaptor primer 1 (AP1, Clontech) and an antisense primer:5′-ATACGGCATCAGGCTTTGGTGCAGTGTCAC-3′ (SEQ ID NO:12)

[0181] corresponding to the 5′ sequence of rRBP1-R (nucleotides741-771). PCR denature was performed at 94° C., 2 minutes; followed by35 amplification cycles of denature at 94° C., 30 seconds; annealing ata temperature of 68° C., 30 seconds and extension at 72° C., 3 minuteswith a final extension 72° C., 7 minutes in a thermal cycler(Perkin-Elmer).

[0182] To examine the expression level of RBP1L1 mRNA, quantitative PCRwas performed on cDNA panels from human normal and cancer tissues (MTCII and Tumor MTC respectively, Clontech). These panels containfirst-strand cDNA from human cancers of the breast, lung, colon, lung,prostate, colon, ovary, and pancreas and from normal human thymus,prostate, testis, ovary, small intestine, colon, and peripheral bloodleukocytes. A set of RBP1L1-specific oligonucleotides: sense =5′-AGGGAATAGCTCGCCAGCAGGTTTTGATG-3′ (SEQ ID NO:13) antisense =5′-TCGGCACTTGTCATATTTTCCAGGTCCGAC-3′; (SEQ ID NO:14)

[0183] spanning a 441-bp cDNA segment was used for PCR with 1 ng of cDNAfrom each tissue. PCR was performed denature at 94° C., 2 minutes;followed by 35 amplification cycles of denature at 94° C., 30 seconds;annealing at temperature of 68° C., 30 seconds and extension at 72° C.,1 minute with a final extension 72° C., 7 minutes. Reaction products (6μl) were evaluated by gel electrophoresis and bands were visualized withethidium bromide. As a loading control, amplified product from a set ofG3PDH control PCR primers (Clontech) was included to assure integrity ofcDNA samples of each tissue.

[0184] After amplifying the cDNA inserts of the immunopositive clones131 and 151 and cloning into a PCRII vector, the inserts were sequenced.The 4032-bp sequence of the two clones was identical. Beyond theβ-galactosidase sequence, each clone contained an open reading frame of107 amino acids ending at a stop codon (TAA) at nucleotides 322-324.Investigation of the premature stop codon by sequencing RT-PCR productsamplified from total RNA from MCF-7 cells and PBMCs, around nucleotide322, showed that none of the 10 cDNA clones examined had a TAA stopcodon at nucleotides 322-324 and that all had the sequence AAA.Consequently, the stop codon likely resulted from a mutation that wasintroduced during the synthesis of MCF-7 cDNA. Correction of thismutation extended the open reading frame an additional 683 nucleotidesand predicted that the full-length protein was 790 amino acids long,consistent with the length of other RB binding proteins.

[0185] 5′ RACE identified additional 1770 nucleotides at the 5′ end ofclone 151 that contained the 5′ UTR and the rest of the 5′ cDNA sequenceof RBP1L1. The complete cDNA sequence (5802 bp) (SEQ ID NO:1) and thepredicted amino acid sequence (1226 amino acids) (SEQ ID NO:2) are shownin FIG. 2. The GenBank accession number of the partial (BCAA) andcomplete RBP1L1 cDNA sequence is AF214114 (SEQ ID NO:15) which encodesthe polypeptide sequence set forth in SEQ ID NO:16.

[0186] NCBI BLAST search results revealed that the following fourprotein sequences are similar to RBP1L1: (1) human RB binding protein(hRBP; accession number AF083249); (2) human RBP1-like protein (hRBP1-L;accession number NP057485); (3) rat RBP1-related protein (rRBP1-R;accession number AF245512); and (4) human RBP1 (HRBP1; accession numberNM002892). Only partial sequences of the RB binding protein andRBP1-like protein gene were available. The derived amino acid sequencealignment of RBP1L1 and hRBP1 is shown in FIG. 3. RBP1L1 shares 74%,72%, 86%, and 37% amino acid sequence identity with hRBP, hRBP1-L,rRBP1-R, and hRBP1, respectively.

EXAMPLE 3 Fusion Protein and Antibody Purification

[0187] GST-RBP1L1 fusion protein (1.5 mg) was purified with theglutathione-argarose beads (Pharmacia Biotech, Alameda, Calif.) from 50mg of total protein extracted from E. coli bacterial lysate. Thepurified fusion protein (2 mg/ml) was coupled to CNBr-activatedSepharose 4B as per the manufacturer's instructions (Pharmacia Biotech).Three milliliters of purified IgG (1.5 mg/ml) was passed over thefusion-protein affinity column, and the column was washed extensivelywith phosphate-buffered saline (PBS) to remove nonspecifically adsorbedproteins. Specifically, bound IgG was eluted with 0.1 M acid glycine atpH 3.0, and the pH was adjusted immediately with 1.0 M Tris buffer (pH9.0).

[0188] Human IgG, isolated from a human hybridoma cell line that was notimmunoreactive to MCF-7 cells was used as a negative control antibody.Control human IgG was prepared by growing human hybridoma cells in RPMImedium 1640 (Life Technologies, Inc., Rockville, Md.) comprising 10%fetal bovine serum until senescence. Conditioned culture medium wasremoved from the cultures, and debris was removed by centrifugation at1,000×g for 10 minutes. IgG was purified with a protein A-Sepharose 4BFast Flow column (Pharmacia Biotech) and eluted with 0.1 M acid glycineat pH 3.0, and the pH was adjusted immediately with 1 M Tris buffer a,pH 9.0. The purified IgG was dialyzed against PBS.

EXAMPLE 4 Carboxyl-Terminal Truncation of Glutathione S-Transferase(GST)-RBP1L1 and Expression of GST Fusion Proteins

[0189] To identify the site of the immunogeneic epitope within the first107 residues of clone 151, a series of carboxyl-terminal deletionconstructs were generated by amplifying and cloning overlapping 5′ DNAfragments of clone 151 (construct 1: 1-105, 2:1-114, 3: 1-142, 4: 1-154,5: 1-208, 6: 1-235, and 7: 1-279) into the GST expression vector, pGEX2.The sense 5′ PCR primer contained a BamHI and all antisense primerscontained an EcoRI site (indicated in bold in the primer sequences shownbelow), allowing amplified products to be cloned into the respectivesites of pGEX-2T. Primers for each of the constructs were as follows: 1:sense = 5′-TGACGGATCCTGCGGCCGCAAAG-3′; (SEQ ID NO:17) antisense =5′-TCTGGAATTCCTTCCCAGAGAGAGGGC-3′; (SEQ ID NO:18) 2: antisense =5′-TCTGGAATTCATTCTTTTTACTTCC-3′; (SEQ ID NO:19) 3: antisense =5′-TCTGGAATTCCCTGATCAGAATGTGTAGG-3′; (SEQ ID NO:20) 4: antisense =5′-TCTGGAATTCCCCAGATTTTCATTGTCTTC-3′; (SEQ ID NO:21) 5: antisense =5′-TCTGGAATTCCCTACCCTAGTTGTGTC-3′; (SEQ ID NO:22) 6: antisense =5′-TCTGGAATTCGCTTTAATCCATTCATC-3′; (SEQ ID NO:23) and 7: antisense =5′-TCTGGAATTCGCTTTTTCTTCCTCAGC-3′; (SEQ ID NO:24)

[0190] Expression of GST-RBP1L1 fusion proteins was induced with 0.1 mMisopropyl β-D-thiogalactopyranoside (IPTG). Bacterial lysates weresubjected to SDS-PAGE and subsequently transferred to nitrocellulosemembranes for Western blot analysis with the purified specific IgGantibody. See Kikumoto, Y., et al. (1995) Hybridoma 14:45-50, which isherein incorporated by reference.

EXAMPLE 5 Synthetic Peptides

[0191] Peptides were synthesized based on the deduced amino acidsequence of the immunoreactive clone and enriched by HPLC to greaterthan 80% purity. (Research Genetics, Inc., Huntsville, Ala.).

[0192] For clone 151, the following eight peptide residues were derivedand synthesized: 1. Residues 29-37 (464-473 of RBP1L1): IKPSLGSKK (SEQID NO:3) 2. Residues 29-38 (464-474 of RBP1L1): IKPSLGSKKN (SEQ IDNO:25) 3. Residues 30-38 (465-474 of RBP1L1): KPSLGSKKN (SEQ ID NO:26)4. Residues 29-36 (464-472 of RBP1L1): IKPSLGSK (SEQ ID NO:27) 5.Residues 30-36 (465-472 of RBP1L1): KPSLGSK (SEQ ID NO:28) 6. Residues31-37 (466-473 of RBP1L1): PSLGSKK (SEQ ID NO:29) 7. Residues 29-35(464-471 of RBP1L1): IKPSLGS (SEQ ID NO:30) 8. Residues 32-38 (467-474of RBP1L1): SLGSKKN (SEQ ID NO:31)

[0193] The C-terminal end of the synthetic peptides contained a carboxylgroup and the N-terminal end contained a free amino group. High pressureliquid chromatography (HPLC) was used to purify these peptides (greaterthan about 80% purity).

EXAMPLE 6 Inhibition of Antibody Activity by Synthetic Peptides

[0194] To determine the minimal number of amino acids necessary forantibody binding, synthetic peptides of various lengths were tested fortheir ability to inhibit the binding of purified specific IgG toGST-RBP1L1 fusion protein by Western blot analysis. The syntheticpeptides tested were: IKPSLGSKK; (SEQ ID NO:3) IKPSLGSKKN; (SEQ IDNO:25) KPSLGSKKN; (SEQ ID NO:26) IKPSLGSK; (SEQ ID NO:27) KPSLGSK; (SEQID NO:28) PSLGSKK; (SEQ ID NO:29) IKPSLGS; (SEQ ID NO:30) SLGSKKN; (SEQID NO:31) and KPSLGSKK. (SEQ ID NO:32)

[0195] The final concentration of each peptide was 100 μg/ml, and theconcentration of purified IgG was 1.5 μg/ml. The antigen and purifiedIgG mixture was agitated at 4° C. overnight, and the inhibition ofantibody binding was assessed by Western blotting using GST-RBP1L1fusion protein as the target antigen.

EXAMPLE 7

[0196] Enzyme-Linked Immunosorbent Assay (ELISA) with Synthetic PeptideAntigens

[0197] The binding of purified specific IgG to synthetic peptides wasassessed by ELISA. 96-well polystyrene plates (Reacti-Bind maleicanhydride-activated plates, Pierce) coated with each of the peptides(100 μl of 5 μg/ml) in PBS, incubated overnight at 4° C., and thenblocked with 1% bovine serum albumin. Purified specific IgG (200 μg/ml)was serially diluted 1:250, 1:500, 1:1000, 1:2000, and 1:4000, thenadded in triplicate to peptide-coated wells (500 ng per well), andincubated for 3 hours at room temperature. Peroxidase-conjugated goatanti-human IgG (Pierce) was added at room temperature for 1 hour,followed by the addition of 400 μg/ml of o-phenylenediaminedihydrochloride in peroxide substrate solution. Reactivity was measuredat 490 nm. Background absorbance without the primary antibody wassubtracted from each sample's absorbance.

EXAMPLE 8 Immunoperoxidase Staining of MCF-7 Cells and PBMCs

[0198] PBMCs were centrifuged onto glass slides using a cytocentrifuge,and MCF-7 cells were plated on slide chambers and incubated in CO₂incubator for 2 days. Cells were fixed in 2% paraformaldehyde. Slideswere dipped in PBS for 5 minutes and then treated sequentially with 0.1%Triton X-100 for 10 minutes to make cell membranes permeable and 3%hydrogen peroxide for 10 minutes to quench endogenous peroxidaseactivity. Next, the slides were washed in running water for 5 minutes,and cells were immunocytochemically stained with VECTASTAIN ABC (Vectorlaboratories, Inc. Burlingame, Calif.). Slides that had been blockedwith goat serum were incubated overnight with either the purifiedRBP1L1-specific IgG (10 μg/ml), a human monoclonal IgG (10 μg/ml) as aMCF-7 negative control, or PBS as a PBMC negative control. After washingwith PBS, the slides were incubated with biotin-labeled goat anti-humanIgG at room temperature for 1 hour. After washing, slides were incubatedwith ABC reagent for 30 minutes, and then added with horseradishperoxidase substrate for color development. The slides werecounterstained with hematoxylin-eosin.

EXAMPLE 9 Expression of RBP1L1 in Human Normal Testis and Cancer Tissues

[0199] Expression of RBP1L1 mRNA in normal human tissues was analyzed byNorthern blotting with RBP1L1-specific probes. Abundant expression of a7.5-kb transcript was detected in the testis, with reduced levels in thethymus, prostate, and ovary. FIG. 4A shows the Northern blot analysis ofRBP1L1 mRNA expression in 13 normal human tissues. Poly(A)+ RNAs (2 μgper lane) were transferred to a nylon membrane and hybridized with³²P-labeled RBP1L1 cDNA probes. Blots were stripped and reprobed with a1.35-kb radiolabeled G3PDH cDNA as a loading control to evaluate theintegrity of RNA. Expression was absent or very low in nine other adulttissues examined. The 1.35-kb G3PDH band was used as a loading control.

[0200] To determine the level of RBP1L1 mRNA expression in a variety ofcancers, a quantitative RT-PCR was performed with a setofRBP1L1-specific primers. Of the six histologic types of cancersexamined, five (breast, ovary, lung, colon, and pancreas) expressedsubstantially higher levels of RBP1L1 mRNA as compared to the six normaltissues. FIG. 4B provides the results of the quantitative reversetranscriptase-polymerase chain reaction (PCR) assay that showsdifferential expression ofRBP1L1 gene. RBP1L1 expression is restrictedto testis as shown by PCR products after 28 PCR cycles (6 μl of productper lane). In contrast, RBP1L1 transcripts are detected in many cancers.G3PDH PCR primers were used as a positive loading control to confirm theintegrity of cDNA. The 983-bp band is the G3PDH PCR product. The 441-bpband is the RBP1L1 PCR product.

[0201] Thus, expression of RBP1L1 in the testis and cancer cells wascomparable. Essentially no expression was detected in prostate cancercells. Therefore, RBP1L1 has a very restricted tissue distribution,being expressed predominately in cancer tissues where it may contributeto the pathophysiology.

EXAMPLE 10 Antigen Epitope Mapping in Recombinant Protein from Clone 151

[0202] Peptide sequences that contain the antigenic epitope wereidentified by testing the GST-RBP1L1 fusion constructs of Example 4.Western blot analysis revealed that the purified IgG recognizedGST-RBP1L1 fusion-constructs 2-7 but not GST-RBP1L1 fusion-construct 1,suggesting that constructs 2-7 contained the antigen epitope. Becauseconstructs 1 and 2 differed by only three residues at thecarboxyl-terminal end, the epitope was expected to be located betweennucleotides 84 and 114 in clone 151 within a 10-amino acid stretch.

[0203] The minimum number of amino acids required for the antibodybinding was determined by testing various truncated synthetic peptidesin ELISAs and on Western blots. The following set of peptides wasgenerated by sequentially reducing the 10-amino acid peptide by oneamino acid from either the amino terminus or the carboxyl terminus:IKPSLGSKK; (SEQ ID NO:3) IKPSLGSKKN; (SEQ ID NO:25) KPSLGSKKN; (SEQ IDNO:26) IKPSLGSK; (SEQ ID NO:27) KPSLGSK; (SEQ ID NO:28) IKPSLGS; (SEQ IDNO:30) KPSLGSKK; (SEQ ID NO:32) and PSLGSKKN. (SEQ ID NO:33)

[0204] The antigenic activity of each peptide (5 μg/ml) was tested forits ability to competitively inhibit the binding of purified specificIgG (0.5 μg/ml) to the GST-RBP1L1 fusion protein on Western blots. Thepeptides IKPSLGSKKN (SEQ ID NO:25) and IKPSLGSKK (SEQ ID NO:3)completely inhibited the purified specific IgG from binding to theGST-RBP1L1 fusion protein from clone 151, indicating that the C-terminalasparagine (N) is not essential for the antibody binding. However,deletion of the C-terminal lysine (K) from IKPSLGSKK (SEQ ID NO:3)substantially reduced the inhibition, and deletion of N-terminalisoleucine (I) from IKPSLGSKK (SEQ ID NO:3) eliminated the inhibition.This result was verified with an ELISA. FIG. 5 shows that RBP1L1specific IgG purified from the serum of a subject with breast cancerbinds to various synthetic peptides in an ELISA. The wells of a 96-wellELISA plate were coated with 100 μl of a peptide (5 μg/ml). The specifichuman IgG (200 μg/ml) was serially diluted from 1:250 to 1:4,000. Thus,IKPSLGSKK (SEQ ID NO:3), residues 465-473 of the full-length RBP1L1protein sequence, represent the minimal epitope recognized by thepurified specific human IgG.

EXAMPLE 11 Cytological and Immunocytochemical Analysis of Antigen

[0205] Purified specific IgG that had been affinity purified on theGST-RBP1L1 fusion-protein affinity column was used to determine thecellular location of the IKPSLGSKK antigen (SEQ ID NO:3) in MCF-7 cellsand in PBMCs. Immunocytochemical staining with the purified specific IgGwas strong in the cytoplasm, with little or no staining of othercellular components. See FIG. 6. FIG. 6A shows staining of the cytoplasmof MCF-7 cells, FIG. 6B shows that no staining of MCF-7 cells wasobserved with the irrelevant human IgG monoclonal antibody alone, FIG.6C shows only membrane staining of human PBMCs with the purified IgG,and FIG. 6D shows membrane staining of PBMCs only with the secondantibody, thereby suggesting that membrane staining is due tonon-specific binding of the second antibody. Thus, antigen-specificstaining was not observed in PBMCs from healthy donors. See FIGS. 6C andD.

EXAMPLE 11 mRNA Microarray

[0206] Analysis of gene expression across multiple tumor samples and ofexpression from tumor and normal tissue of the same subject wasperformed on Cancer Profiling Expression Array on nylon membrane thattogether contain more than 241 cDNA pairs, each pair representing tumorand normal tissue from a single subject. These arrays provide ahigh-throughput format for analyzing differential gene expression(Clontech). The microarray contains cDNA from normal and carcinoma ofbreast, uterus, colon, stomach, ovary, lung, kidney, rectum, thyroidgland, cervix, small intestine, pancreas, and prostate. The legend tothe microarray is shown in FIG. 7A.

[0207] Membranes were hybridized with a ³²P-labeled 441-bp fragment ofRBP1L1 cDNA that was amplified by PCR from the immunopositive clone 151.Blots were washed twice in 0.1% standard saline citrate (SSC) and 0.1 %SDS for 20 minutes and then exposed to Kodak BIOMAX film at −80 ° C.with an intensifying screen for 24 hours. As a loading control, blotswere stripped and reprobed with the control ubiquitin cDNA (Clontech) toprove equal quantity of cDNA distribution in each pair of normal andcancer tissue. See Zhumabayeva and Adhikari (2001) “Cancer ProfilingArray: A New Approach for Evaluating Differential Gene Expressions inVarious Cancer Tissues” CLONTECHniques (Clontech), which is hereinincorporated by reference. See FIG. 7B.

[0208] The cancer profiling array shows the tissue specific expressionof RBP1L1. The cancer profiling array was hybridized separately with aradiolabeled RBP1L1 probe for RBP1L1, as shown in panel A, and aradiolabeled probe for the housekeeping gene ubiquitin, as shown inpanel B. Numbers indicate tissue types in columns as follows: 1=breast,2=uterus, 3=colon, 4=stomach, 5=ovary, 6=lung, 7=kidney, 8=rectum,9=thyroid gland, 10=cervix, 11=small intestine, 12=pancreas, and13=prostate. N=normal, T=tumor, Ubi=ubiquitin cDNA, cc=cancer cell linecDNAs.

[0209] To the extent necessary to understand or complete the disclosureof the present invention, all publications, patents, and patentapplications mentioned herein are expressly incorporated by referencetherein to the same extent as though each were individually soincorporated.

[0210] Having thus described exemplary embodiments of the presentinvention, it should be noted by those skilled in the art that thewithin disclosures are exemplary only and that various otheralternatives, adaptations, and modifications may be made within thescope of the present invention. Accordingly, the present invention isnot limited to the specific embodiments as illustrated herein, but isonly limited by the following claims.

1 33 1 5802 DNA Homo sapiens CDS (463)..(4143) 1 cgatgtttgc ccgtcagtcgagtccggagt gaggagctcg gtcgccgaag cggagggaga 60 ctcttgagct tcatcttgccgccgccacgg ccaccgcctg gacctttgcc cggagggagc 120 tgcagagggt ccatcgccgccgtcctctgg agggcagcgc gattgggggc ccggacctcc 180 agtccggggg ggatttttcgtcgtccccct ccccccaacc agggagcccg agcggccgcc 240 aaacaaaggt accagtcgccgccgcgggag gaggaggagc cggagcctct gcctcacagc 300 cgctggaccc gccgccttcttccccatctc tcccccgggc ctgctggttt tgggggggag 360 aaggagagag gggactctggacgtgccagg gtcaaatctc gcctccgagg aaggtgcaac 420 tgaacctggt gttttaaaggataccttggt cccaaagtca tc atg aag gcc ctt 474 Met Lys Ala Leu 1 gat gagcct ccc tat ttg aca gtg ggc act gat gtg agt gct aaa tac 522 Asp Glu ProPro Tyr Leu Thr Val Gly Thr Asp Val Ser Ala Lys Tyr 5 10 15 20 aga ggagcc ttt tgt gaa gcc aag atc aag aca gca aaa aga ctt gtc 570 Arg Gly AlaPhe Cys Glu Ala Lys Ile Lys Thr Ala Lys Arg Leu Val 25 30 35 aaa gtc aaggtg aca ttt aga cat gat tct tca aca gtg gaa gtt cag 618 Lys Val Lys ValThr Phe Arg His Asp Ser Ser Thr Val Glu Val Gln 40 45 50 gat gac cac ataaag ggc cca cta aag gta gga gct att gtg gaa gtg 666 Asp Asp His Ile LysGly Pro Leu Lys Val Gly Ala Ile Val Glu Val 55 60 65 aag aat ctt gat ggtgca tat cag gaa gct gtt atc aat aaa cta aca 714 Lys Asn Leu Asp Gly AlaTyr Gln Glu Ala Val Ile Asn Lys Leu Thr 70 75 80 gat gcg agt tgg tac actgta gtt ttt gat gac gga gat gag aag aca 762 Asp Ala Ser Trp Tyr Thr ValVal Phe Asp Asp Gly Asp Glu Lys Thr 85 90 95 100 ctg aga cga tct tca ctgtgc ctg aaa gga gag agg cat ttt gcc gaa 810 Leu Arg Arg Ser Ser Leu CysLeu Lys Gly Glu Arg His Phe Ala Glu 105 110 115 agt gaa aca tta gac cagctc cca ctc acc aac cct gag cat ttt ggc 858 Ser Glu Thr Leu Asp Gln LeuPro Leu Thr Asn Pro Glu His Phe Gly 120 125 130 act cca gtc ata gga aagaaa aca aat aga gga aga aga tct aat cat 906 Thr Pro Val Ile Gly Lys LysThr Asn Arg Gly Arg Arg Ser Asn His 135 140 145 ata cca gag gaa gag tcttca tca tcc tcc agt gat gaa gat gag gat 954 Ile Pro Glu Glu Glu Ser SerSer Ser Ser Ser Asp Glu Asp Glu Asp 150 155 160 gat agg aaa cag att gatgag cta cta ggc aaa gtt gta tgt gta gat 1002 Asp Arg Lys Gln Ile Asp GluLeu Leu Gly Lys Val Val Cys Val Asp 165 170 175 180 tac att agt ttg gataaa aag aaa gca ctg tgg ttt cct gca ttg gtg 1050 Tyr Ile Ser Leu Asp LysLys Lys Ala Leu Trp Phe Pro Ala Leu Val 185 190 195 gtt tgt cct gat tgtagt gat gag att gct gta aaa aag gac aat att 1098 Val Cys Pro Asp Cys SerAsp Glu Ile Ala Val Lys Lys Asp Asn Ile 200 205 210 ctt gtt cga tct ttcaaa gat gga aaa ttt act tca gtt cca aga aaa 1146 Leu Val Arg Ser Phe LysAsp Gly Lys Phe Thr Ser Val Pro Arg Lys 215 220 225 gat gtc cat gaa attact agt gac act gca cca aag cct gat gct gtt 1194 Asp Val His Glu Ile ThrSer Asp Thr Ala Pro Lys Pro Asp Ala Val 230 235 240 tta aag caa gcc tttgaa cag gca ctt gaa ttt cac aaa agt aga act 1242 Leu Lys Gln Ala Phe GluGln Ala Leu Glu Phe His Lys Ser Arg Thr 245 250 255 260 att cct gct aactgg aag act gaa ttg aaa gaa gat agc tct agc agt 1290 Ile Pro Ala Asn TrpLys Thr Glu Leu Lys Glu Asp Ser Ser Ser Ser 265 270 275 gaa gca gag gaagaa gag gag gag gaa gat gat gaa aaa gaa aag gag 1338 Glu Ala Glu Glu GluGlu Glu Glu Glu Asp Asp Glu Lys Glu Lys Glu 280 285 290 gat aat agc agtgaa gaa gaa gaa gaa ata gaa cca ttt cca gaa gaa 1386 Asp Asn Ser Ser GluGlu Glu Glu Glu Ile Glu Pro Phe Pro Glu Glu 295 300 305 agg gag aac tttctt cag caa ttg tac aaa ttt atg gaa gat aga ggt 1434 Arg Glu Asn Phe LeuGln Gln Leu Tyr Lys Phe Met Glu Asp Arg Gly 310 315 320 aca cct att aacaaa caa cct gta ctt gga tat cga aat ttg aat ctc 1482 Thr Pro Ile Asn LysGln Pro Val Leu Gly Tyr Arg Asn Leu Asn Leu 325 330 335 340 ttt aag ttattc aga ctt gta cac aaa ctt gga gga ttt gat aat att 1530 Phe Lys Leu PheArg Leu Val His Lys Leu Gly Gly Phe Asp Asn Ile 345 350 355 gaa agt ggagct gtt tgg aaa caa gtc tac caa gat ctt gga atc cct 1578 Glu Ser Gly AlaVal Trp Lys Gln Val Tyr Gln Asp Leu Gly Ile Pro 360 365 370 gtc tta aattca gct gca gga tac aat gtt aaa tgt gct tat aaa aaa 1626 Val Leu Asn SerAla Ala Gly Tyr Asn Val Lys Cys Ala Tyr Lys Lys 375 380 385 tac tta tatggt ttt gag gag tac tgt aga tca gcc aac att gaa ttt 1674 Tyr Leu Tyr GlyPhe Glu Glu Tyr Cys Arg Ser Ala Asn Ile Glu Phe 390 395 400 cag atg gcattg cca gag aaa gtt gtt aac aag caa tgt aag gag tgt 1722 Gln Met Ala LeuPro Glu Lys Val Val Asn Lys Gln Cys Lys Glu Cys 405 410 415 420 gaa aatgta aaa gaa ata aaa gtt aag gag gaa aat gaa aca gag atc 1770 Glu Asn ValLys Glu Ile Lys Val Lys Glu Glu Asn Glu Thr Glu Ile 425 430 435 aaa gaaata aag atg gag gag gag agg aat ata ata cca aga gaa gaa 1818 Lys Glu IleLys Met Glu Glu Glu Arg Asn Ile Ile Pro Arg Glu Glu 440 445 450 aag cctatt gag gat gaa att gaa aga aaa gaa aat att aag ccc tct 1866 Lys Pro IleGlu Asp Glu Ile Glu Arg Lys Glu Asn Ile Lys Pro Ser 455 460 465 ctg ggaagt aaa aag aat tta tta gaa tct ata cct aca cat tct gat 1914 Leu Gly SerLys Lys Asn Leu Leu Glu Ser Ile Pro Thr His Ser Asp 470 475 480 cag gaaaaa gaa gtt aac att aaa aaa cca gaa gac aat gaa aat ctg 1962 Gln Glu LysGlu Val Asn Ile Lys Lys Pro Glu Asp Asn Glu Asn Leu 485 490 495 500 gatgac aaa gat gat gac aca act agg gta gat gaa tcc ctc aac ata 2010 Asp AspLys Asp Asp Asp Thr Thr Arg Val Asp Glu Ser Leu Asn Ile 505 510 515 aaggta gaa gct gag gaa gaa aaa gca aaa tct gga tac gat gaa tgg 2058 Lys ValGlu Ala Glu Glu Glu Lys Ala Lys Ser Gly Tyr Asp Glu Trp 520 525 530 attaaa gca gat aaa ata gta aga cct gct gat aaa aat gtg cca aag 2106 Ile LysAla Asp Lys Ile Val Arg Pro Ala Asp Lys Asn Val Pro Lys 535 540 545 ataaaa cat cgg aag aaa ata aag aat aaa tta gac aaa gaa aaa gac 2154 Ile LysHis Arg Lys Lys Ile Lys Asn Lys Leu Asp Lys Glu Lys Asp 550 555 560 aaagat gaa aaa tac tct cca aaa aac tgt aaa ctt cgg cgc ttg tcc 2202 Lys AspGlu Lys Tyr Ser Pro Lys Asn Cys Lys Leu Arg Arg Leu Ser 565 570 575 580aaa cca cca ttt cag aca aat cca tct cct gaa atg gta tcc aaa ctg 2250 LysPro Pro Phe Gln Thr Asn Pro Ser Pro Glu Met Val Ser Lys Leu 585 590 595gat ctc act gat gcc aaa aac tct gat act gct cat att aag tcc ata 2298 AspLeu Thr Asp Ala Lys Asn Ser Asp Thr Ala His Ile Lys Ser Ile 600 605 610gaa att act tcg atc ctt aat gga ctt caa gct tct gaa agt tct gct 2346 GluIle Thr Ser Ile Leu Asn Gly Leu Gln Ala Ser Glu Ser Ser Ala 615 620 625gaa gac agt gag cag gaa gat gag aga ggt gct caa gac atg gat aat 2394 GluAsp Ser Glu Gln Glu Asp Glu Arg Gly Ala Gln Asp Met Asp Asn 630 635 640aat ggc aaa gaa gaa tct aag att gat cat ttg acc aac aac aga aat 2442 AsnGly Lys Glu Glu Ser Lys Ile Asp His Leu Thr Asn Asn Arg Asn 645 650 655660 gat ctt att tca aag gag gaa cag aac agt tca tct ttg cta gaa gaa 2490Asp Leu Ile Ser Lys Glu Glu Gln Asn Ser Ser Ser Leu Leu Glu Glu 665 670675 aac aaa gtt cat gca gat ttg gta ata tcc aaa cca gtg tca aaa tct 2538Asn Lys Val His Ala Asp Leu Val Ile Ser Lys Pro Val Ser Lys Ser 680 685690 cca gaa aga tta agg aaa gat ata gaa gta tta tcc gaa gat act gat 2586Pro Glu Arg Leu Arg Lys Asp Ile Glu Val Leu Ser Glu Asp Thr Asp 695 700705 tat gaa gaa gat gaa gtc aca aaa aag aga aag gat gtc aag aag gac 2634Tyr Glu Glu Asp Glu Val Thr Lys Lys Arg Lys Asp Val Lys Lys Asp 710 715720 aca aca gat aaa tct tca aaa cca caa ata aaa cgt ggt aaa aga agg 2682Thr Thr Asp Lys Ser Ser Lys Pro Gln Ile Lys Arg Gly Lys Arg Arg 725 730735 740 tat tgc aat aca gaa gag tgt cta aaa act gga tca cct ggc aaa aag2730 Tyr Cys Asn Thr Glu Glu Cys Leu Lys Thr Gly Ser Pro Gly Lys Lys 745750 755 gaa gag aag gcc aag aac aaa gaa tca ctt tgc atg gaa aac agt agc2778 Glu Glu Lys Ala Lys Asn Lys Glu Ser Leu Cys Met Glu Asn Ser Ser 760765 770 aac agc tct tca gat gaa gat gaa gaa gaa aca aaa gca aag atg aca2826 Asn Ser Ser Ser Asp Glu Asp Glu Glu Glu Thr Lys Ala Lys Met Thr 775780 785 cca act aag aaa tac aat ggt ttg gag gaa aaa aga aaa tct cta cgg2874 Pro Thr Lys Lys Tyr Asn Gly Leu Glu Glu Lys Arg Lys Ser Leu Arg 790795 800 aca act ggt ttc tat tca gga ttt tca gaa gtg gca gaa aaa agg att2922 Thr Thr Gly Phe Tyr Ser Gly Phe Ser Glu Val Ala Glu Lys Arg Ile 805810 815 820 aaa ctt tta aat aac tct gat gaa aga ctt caa aac agc agg gccaaa 2970 Lys Leu Leu Asn Asn Ser Asp Glu Arg Leu Gln Asn Ser Arg Ala Lys825 830 835 gat cga aaa gat gtc tgg tca agt att cag gga cag tgg cct aaaaaa 3018 Asp Arg Lys Asp Val Trp Ser Ser Ile Gln Gly Gln Trp Pro Lys Lys840 845 850 acg ctg aaa gag ctt ttt tca gac tct gat act gag gct gca gcttcc 3066 Thr Leu Lys Glu Leu Phe Ser Asp Ser Asp Thr Glu Ala Ala Ala Ser855 860 865 cca ccg cat cct gcc cca gag gag ggg gtg gca gag gag tca ctgcag 3114 Pro Pro His Pro Ala Pro Glu Glu Gly Val Ala Glu Glu Ser Leu Gln870 875 880 act gtg gct gaa gag gag agt tgt tca ccc agt gta gaa cta gaaaaa 3162 Thr Val Ala Glu Glu Glu Ser Cys Ser Pro Ser Val Glu Leu Glu Lys885 890 895 900 cca cct cca gtc aat gtc gat agt aaa ccc att gaa gaa gaaaca gta 3210 Pro Pro Pro Val Asn Val Asp Ser Lys Pro Ile Glu Glu Glu ThrVal 905 910 915 gag gtc aat gac aga aaa gca gaa ttt cca agt agt ggc agtaat tca 3258 Glu Val Asn Asp Arg Lys Ala Glu Phe Pro Ser Ser Gly Ser AsnSer 920 925 930 gtg cta aat acc cct cct act aca cct gaa tcg cct tca tcagtc act 3306 Val Leu Asn Thr Pro Pro Thr Thr Pro Glu Ser Pro Ser Ser ValThr 935 940 945 gta aca gaa ggc agc cgg cag cag tct tct gta aca gta tcagaa cca 3354 Val Thr Glu Gly Ser Arg Gln Gln Ser Ser Val Thr Val Ser GluPro 950 955 960 ctg gct cca aac caa gaa gag gtt cga agt atc aag agt gaaact gat 3402 Leu Ala Pro Asn Gln Glu Glu Val Arg Ser Ile Lys Ser Glu ThrAsp 965 970 975 980 agc aca att gag gtg gat agt gtt gct ggg gag ctc caagac ctc cag 3450 Ser Thr Ile Glu Val Asp Ser Val Ala Gly Glu Leu Gln AspLeu Gln 985 990 995 tct gaa ggg aat agc tcg cca gca ggt ttt gat gcc agtgtg agc tca 3498 Ser Glu Gly Asn Ser Ser Pro Ala Gly Phe Asp Ala Ser ValSer Ser 1000 1005 1010 agc agt agt aat cag cca gaa cca gaa cat cct gaaaaa gcc tgt aca 3546 Ser Ser Ser Asn Gln Pro Glu Pro Glu His Pro Glu LysAla Cys Thr 1015 1020 1025 ggt cag aaa aga gtg aaa gat gct cag gga ggagga agt tca tca aaa 3594 Gly Gln Lys Arg Val Lys Asp Ala Gln Gly Gly GlySer Ser Ser Lys 1030 1035 1040 aag cag aaa aga agc cat aaa gca aca gtggta aac aac aaa aag aag 3642 Lys Gln Lys Arg Ser His Lys Ala Thr Val ValAsn Asn Lys Lys Lys 1045 1050 1055 1060 gga aaa ggc aca aat agt agt gatagt gaa gaa ctt tca gct ggt gaa 3690 Gly Lys Gly Thr Asn Ser Ser Asp SerGlu Glu Leu Ser Ala Gly Glu 1065 1070 1075 agt ata act aag agt cag ccagtc aaa tca gtt tcc act gga atg aag 3738 Ser Ile Thr Lys Ser Gln Pro ValLys Ser Val Ser Thr Gly Met Lys 1080 1085 1090 tct cat agt acc aaa tctccc gca agg acg cag tct cca gga aaa tgt 3786 Ser His Ser Thr Lys Ser ProAla Arg Thr Gln Ser Pro Gly Lys Cys 1095 1100 1105 gga aag aat ggt gataag gat cct gat ctc aag gaa ccc agt aat cga 3834 Gly Lys Asn Gly Asp LysAsp Pro Asp Leu Lys Glu Pro Ser Asn Arg 1110 1115 1120 tta ccc aaa gtttac aaa tgg agt ttt cag atg tcg gac ctg gaa aat 3882 Leu Pro Lys Val TyrLys Trp Ser Phe Gln Met Ser Asp Leu Glu Asn 1125 1130 1135 1140 atg acaagt gcc gaa cgc atc aca att ctt caa gaa aaa ctt caa gaa 3930 Met Thr SerAla Glu Arg Ile Thr Ile Leu Gln Glu Lys Leu Gln Glu 1145 1150 1155 atcaga aaa cat tat ctg tca tta aaa tct gaa gta gct tcc att gat 3978 Ile ArgLys His Tyr Leu Ser Leu Lys Ser Glu Val Ala Ser Ile Asp 1160 1165 1170cgg agg aga aag cgt tta aag aag aaa gag aga gaa agt gct gct aca 4026 ArgArg Arg Lys Arg Leu Lys Lys Lys Glu Arg Glu Ser Ala Ala Thr 1175 11801185 tcc tca tcc tcc tct tca cct tca tcc agc tcc ata aca gct gct gct4074 Ser Ser Ser Ser Ser Ser Pro Ser Ser Ser Ser Ile Thr Ala Ala Ala1190 1195 1200 atg tta act tta gct gaa ccg tca atg tcc agc gca tca caaaat gga 4122 Met Leu Thr Leu Ala Glu Pro Ser Met Ser Ser Ala Ser Gln AsnGly 1205 1210 1215 1220 atg tca gtt gag tgc agg tga cagcaggacttgctaaggca ctttgcactt 4173 Met Ser Val Glu Cys Arg 1225 aatggctgttgagggccact ttttttttat actgcacagt ggcacaaaaa aatatcagac 4233 aagcactattttatatttaa aaattgtttc ttgacaagcc gacttggcac ttaagtgcac 4293 ttttgtatgaagaaaagtac aatgaactgc ttttcctcaa gcaataattg tttccaactt 4353 gtctgggaattgtgtgtctg gtaactggaa ggccttccac tgtggcaaat ggaggcttct 4413 cactgcctgtagagacaata cagtaagcat agttaagggg tgggtcagaa catgttaaga 4473 taacttactgtatatgtatt cccttgtatt ttgttaaagc tggaacattt gatatttttc 4533 catttatttatgaaaaaata tgaacctatt ttcatttgta caaggtaatt gttttttaaa 4593 gcaagtcaccttagggtggc tttaattgta taagtcaagc acatgtaata aattcaaaac 4653 ctgcagttaacaggatatta gacattaatc ctggtaacca aatattaaag attctcttta 4713 aaaaagactgaacatgttta caggtttgaa ttaggctaaa aggtcttgca gtggcttttc 4773 atggcccttcaaattggaat ggaactactg tactttgcca tttttctata aatcagtatt 4833 tttttttaattttgatatac attgtgtgaa aaaagaaaat ggctaataaa ctgtattaaa 4893 tcttaaacaatgtataaaga ttgtacttag ccagttcaaa ggtatattta ttcataatga 4953 attataacagttatattttt gtgttttctt gtaaatgttt cttttccctt aaatacagat 5013 aattcatttgtattgcttat tttattatga gctacaacaa aaggacttca ggaacaagta 5073 atgtattagtatggttcaag attgttgata ggaactgtct caaaaggatg gtggttattt 5133 taaatataaatagctaatgg gggtggtagg cctataaaat taaatgcctt gtataagatc 5193 caaaatgaatgcaaaattgt tttcacttgt attgacttta tgttgtatga ttccaatctc 5253 tgttctgtttggcacttgta tttaattctt cacctttgta agacatttgt atattgtgga 5313 tgtgttcattcaagctattt aatatctggc actgttaata cacagtactt tattgtacag 5373 actgttttactgttttaatt gtagttctgt gtactttttt tggatggggc tggcatgttt 5433 tctttgtttcctggcaatac gacgtgggaa tttcaatgcg ttttgttgta gatgctaacg 5493 tgtcagaatcctttacattc aacttttcta agaaaagcat tttcagtctt gtagtgtgtg 5553 cttacagtaactaattttgt tgaaaatggt ttcaagttat tcaaatttgt acaggactgt 5613 aaagatttgttgacagcaaa atgttgaaga aaaaagctta tagaataaaa gctataaagt 5673 atatattaggatctgcaaac aatgaagaat tatgtaatat attgtacaaa tgtaaagcaa 5733 aggctttgaaataaaatgcc attgtttgtg aatccttaaa aaaaaaaaaa aaaaaaaaaa 5793 aaaaaaaaa5802 2 1226 PRT Homo sapiens 2 Met Lys Ala Leu Asp Glu Pro Pro Tyr LeuThr Val Gly Thr Asp Val 1 5 10 15 Ser Ala Lys Tyr Arg Gly Ala Phe CysGlu Ala Lys Ile Lys Thr Ala 20 25 30 Lys Arg Leu Val Lys Val Lys Val ThrPhe Arg His Asp Ser Ser Thr 35 40 45 Val Glu Val Gln Asp Asp His Ile LysGly Pro Leu Lys Val Gly Ala 50 55 60 Ile Val Glu Val Lys Asn Leu Asp GlyAla Tyr Gln Glu Ala Val Ile 65 70 75 80 Asn Lys Leu Thr Asp Ala Ser TrpTyr Thr Val Val Phe Asp Asp Gly 85 90 95 Asp Glu Lys Thr Leu Arg Arg SerSer Leu Cys Leu Lys Gly Glu Arg 100 105 110 His Phe Ala Glu Ser Glu ThrLeu Asp Gln Leu Pro Leu Thr Asn Pro 115 120 125 Glu His Phe Gly Thr ProVal Ile Gly Lys Lys Thr Asn Arg Gly Arg 130 135 140 Arg Ser Asn His IlePro Glu Glu Glu Ser Ser Ser Ser Ser Ser Asp 145 150 155 160 Glu Asp GluAsp Asp Arg Lys Gln Ile Asp Glu Leu Leu Gly Lys Val 165 170 175 Val CysVal Asp Tyr Ile Ser Leu Asp Lys Lys Lys Ala Leu Trp Phe 180 185 190 ProAla Leu Val Val Cys Pro Asp Cys Ser Asp Glu Ile Ala Val Lys 195 200 205Lys Asp Asn Ile Leu Val Arg Ser Phe Lys Asp Gly Lys Phe Thr Ser 210 215220 Val Pro Arg Lys Asp Val His Glu Ile Thr Ser Asp Thr Ala Pro Lys 225230 235 240 Pro Asp Ala Val Leu Lys Gln Ala Phe Glu Gln Ala Leu Glu PheHis 245 250 255 Lys Ser Arg Thr Ile Pro Ala Asn Trp Lys Thr Glu Leu LysGlu Asp 260 265 270 Ser Ser Ser Ser Glu Ala Glu Glu Glu Glu Glu Glu GluAsp Asp Glu 275 280 285 Lys Glu Lys Glu Asp Asn Ser Ser Glu Glu Glu GluGlu Ile Glu Pro 290 295 300 Phe Pro Glu Glu Arg Glu Asn Phe Leu Gln GlnLeu Tyr Lys Phe Met 305 310 315 320 Glu Asp Arg Gly Thr Pro Ile Asn LysGln Pro Val Leu Gly Tyr Arg 325 330 335 Asn Leu Asn Leu Phe Lys Leu PheArg Leu Val His Lys Leu Gly Gly 340 345 350 Phe Asp Asn Ile Glu Ser GlyAla Val Trp Lys Gln Val Tyr Gln Asp 355 360 365 Leu Gly Ile Pro Val LeuAsn Ser Ala Ala Gly Tyr Asn Val Lys Cys 370 375 380 Ala Tyr Lys Lys TyrLeu Tyr Gly Phe Glu Glu Tyr Cys Arg Ser Ala 385 390 395 400 Asn Ile GluPhe Gln Met Ala Leu Pro Glu Lys Val Val Asn Lys Gln 405 410 415 Cys LysGlu Cys Glu Asn Val Lys Glu Ile Lys Val Lys Glu Glu Asn 420 425 430 GluThr Glu Ile Lys Glu Ile Lys Met Glu Glu Glu Arg Asn Ile Ile 435 440 445Pro Arg Glu Glu Lys Pro Ile Glu Asp Glu Ile Glu Arg Lys Glu Asn 450 455460 Ile Lys Pro Ser Leu Gly Ser Lys Lys Asn Leu Leu Glu Ser Ile Pro 465470 475 480 Thr His Ser Asp Gln Glu Lys Glu Val Asn Ile Lys Lys Pro GluAsp 485 490 495 Asn Glu Asn Leu Asp Asp Lys Asp Asp Asp Thr Thr Arg ValAsp Glu 500 505 510 Ser Leu Asn Ile Lys Val Glu Ala Glu Glu Glu Lys AlaLys Ser Gly 515 520 525 Tyr Asp Glu Trp Ile Lys Ala Asp Lys Ile Val ArgPro Ala Asp Lys 530 535 540 Asn Val Pro Lys Ile Lys His Arg Lys Lys IleLys Asn Lys Leu Asp 545 550 555 560 Lys Glu Lys Asp Lys Asp Glu Lys TyrSer Pro Lys Asn Cys Lys Leu 565 570 575 Arg Arg Leu Ser Lys Pro Pro PheGln Thr Asn Pro Ser Pro Glu Met 580 585 590 Val Ser Lys Leu Asp Leu ThrAsp Ala Lys Asn Ser Asp Thr Ala His 595 600 605 Ile Lys Ser Ile Glu IleThr Ser Ile Leu Asn Gly Leu Gln Ala Ser 610 615 620 Glu Ser Ser Ala GluAsp Ser Glu Gln Glu Asp Glu Arg Gly Ala Gln 625 630 635 640 Asp Met AspAsn Asn Gly Lys Glu Glu Ser Lys Ile Asp His Leu Thr 645 650 655 Asn AsnArg Asn Asp Leu Ile Ser Lys Glu Glu Gln Asn Ser Ser Ser 660 665 670 LeuLeu Glu Glu Asn Lys Val His Ala Asp Leu Val Ile Ser Lys Pro 675 680 685Val Ser Lys Ser Pro Glu Arg Leu Arg Lys Asp Ile Glu Val Leu Ser 690 695700 Glu Asp Thr Asp Tyr Glu Glu Asp Glu Val Thr Lys Lys Arg Lys Asp 705710 715 720 Val Lys Lys Asp Thr Thr Asp Lys Ser Ser Lys Pro Gln Ile LysArg 725 730 735 Gly Lys Arg Arg Tyr Cys Asn Thr Glu Glu Cys Leu Lys ThrGly Ser 740 745 750 Pro Gly Lys Lys Glu Glu Lys Ala Lys Asn Lys Glu SerLeu Cys Met 755 760 765 Glu Asn Ser Ser Asn Ser Ser Ser Asp Glu Asp GluGlu Glu Thr Lys 770 775 780 Ala Lys Met Thr Pro Thr Lys Lys Tyr Asn GlyLeu Glu Glu Lys Arg 785 790 795 800 Lys Ser Leu Arg Thr Thr Gly Phe TyrSer Gly Phe Ser Glu Val Ala 805 810 815 Glu Lys Arg Ile Lys Leu Leu AsnAsn Ser Asp Glu Arg Leu Gln Asn 820 825 830 Ser Arg Ala Lys Asp Arg LysAsp Val Trp Ser Ser Ile Gln Gly Gln 835 840 845 Trp Pro Lys Lys Thr LeuLys Glu Leu Phe Ser Asp Ser Asp Thr Glu 850 855 860 Ala Ala Ala Ser ProPro His Pro Ala Pro Glu Glu Gly Val Ala Glu 865 870 875 880 Glu Ser LeuGln Thr Val Ala Glu Glu Glu Ser Cys Ser Pro Ser Val 885 890 895 Glu LeuGlu Lys Pro Pro Pro Val Asn Val Asp Ser Lys Pro Ile Glu 900 905 910 GluGlu Thr Val Glu Val Asn Asp Arg Lys Ala Glu Phe Pro Ser Ser 915 920 925Gly Ser Asn Ser Val Leu Asn Thr Pro Pro Thr Thr Pro Glu Ser Pro 930 935940 Ser Ser Val Thr Val Thr Glu Gly Ser Arg Gln Gln Ser Ser Val Thr 945950 955 960 Val Ser Glu Pro Leu Ala Pro Asn Gln Glu Glu Val Arg Ser IleLys 965 970 975 Ser Glu Thr Asp Ser Thr Ile Glu Val Asp Ser Val Ala GlyGlu Leu 980 985 990 Gln Asp Leu Gln Ser Glu Gly Asn Ser Ser Pro Ala GlyPhe Asp Ala 995 1000 1005 Ser Val Ser Ser Ser Ser Ser Asn Gln Pro GluPro Glu His Pro Glu 1010 1015 1020 Lys Ala Cys Thr Gly Gln Lys Arg ValLys Asp Ala Gln Gly Gly Gly 1025 1030 1035 1040 Ser Ser Ser Lys Lys GlnLys Arg Ser His Lys Ala Thr Val Val Asn 1045 1050 1055 Asn Lys Lys LysGly Lys Gly Thr Asn Ser Ser Asp Ser Glu Glu Leu 1060 1065 1070 Ser AlaGly Glu Ser Ile Thr Lys Ser Gln Pro Val Lys Ser Val Ser 1075 1080 1085Thr Gly Met Lys Ser His Ser Thr Lys Ser Pro Ala Arg Thr Gln Ser 10901095 1100 Pro Gly Lys Cys Gly Lys Asn Gly Asp Lys Asp Pro Asp Leu LysGlu 1105 1110 1115 1120 Pro Ser Asn Arg Leu Pro Lys Val Tyr Lys Trp SerPhe Gln Met Ser 1125 1130 1135 Asp Leu Glu Asn Met Thr Ser Ala Glu ArgIle Thr Ile Leu Gln Glu 1140 1145 1150 Lys Leu Gln Glu Ile Arg Lys HisTyr Leu Ser Leu Lys Ser Glu Val 1155 1160 1165 Ala Ser Ile Asp Arg ArgArg Lys Arg Leu Lys Lys Lys Glu Arg Glu 1170 1175 1180 Ser Ala Ala ThrSer Ser Ser Ser Ser Ser Pro Ser Ser Ser Ser Ile 1185 1190 1195 1200 ThrAla Ala Ala Met Leu Thr Leu Ala Glu Pro Ser Met Ser Ser Ala 1205 12101215 Ser Gln Asn Gly Met Ser Val Glu Cys Arg 1220 1225 3 9 PRT Homosapiens 3 Ile Lys Pro Ser Leu Gly Ser Lys Lys 1 5 4 1255 PRT Homosapiens 4 Met Lys Ala Ala Asp Glu Pro Ala Tyr Leu Thr Val Gly Thr AspVal 1 5 10 15 Ser Ala Lys Tyr Arg Gly Ala Phe Cys Glu Ala Lys Ile LysThr Val 20 25 30 Lys Arg Leu Val Lys Val Lys Val Leu Leu Lys Gln Asp AsnThr Thr 35 40 45 Gln Leu Val Gln Asp Asp Gln Val Lys Gly Pro Leu Arg ValGly Ala 50 55 60 Ile Val Glu Thr Arg Thr Ser Asp Gly Ser Phe Gln Glu AlaIle Ile 65 70 75 80 Ser Lys Leu Thr Asp Ala Ser Trp Tyr Thr Val Val PheAsp Asp Gly 85 90 95 Asp Glu Arg Thr Leu Arg Arg Thr Ser Leu Cys Leu LysGly Glu Arg 100 105 110 His Phe Ala Glu Ser Glu Thr Leu Asp Gln Leu ProLeu Thr Asn Pro 115 120 125 Glu His Phe Gly Thr Pro Val Ile Ala Lys LysThr Asn Arg Gly Arg 130 135 140 Arg Ser Ser Leu Pro Val Thr Glu Asp GluLys Glu Glu Glu Ser Ser 145 150 155 160 Glu Glu Glu Asp Glu Asp Lys ArgArg Leu Asn Asp Glu Leu Leu Gly 165 170 175 Lys Val Val Ser Val Val SerAla Thr Glu Arg Thr Glu Trp Tyr Pro 180 185 190 Ala Leu Val Ile Ser ProSer Cys Asn Asp Asp Ile Thr Val Lys Lys 195 200 205 Asp Gln Cys Leu ValArg Ser Phe Ile Asp Ser Lys Phe Tyr Ser Ile 210 215 220 Ala Arg Lys AspIle Lys Glu Val Asp Ile Leu Asn Leu Pro Glu Ser 225 230 235 240 Glu LeuSer Thr Lys Pro Gly Leu Gln Lys Ala Ser Ile Phe Leu Lys 245 250 255 ThrArg Val Val Pro Asp Asn Trp Lys Met Asp Ile Ser Glu Ile Leu 260 265 270Glu Ser Ser Ser Ser Asp Asp Glu Asp Gly Pro Ala Glu Glu Asn Asp 275 280285 Glu Glu Lys Glu Lys Glu Ala Lys Lys Thr Glu Glu Glu Val Pro Glu 290295 300 Glu Glu Leu Asp Pro Glu Glu Arg Asp Asn Phe Leu Gln Gln Leu Tyr305 310 315 320 Lys Phe Met Glu Asp Arg Gly Thr Pro Ile Asn Lys Pro ProVal Leu 325 330 335 Gly Tyr Lys Asp Leu Asn Leu Phe Lys Leu Phe Arg LeuVal Tyr His 340 345 350 Gln Gly Gly Cys Asp Asn Ile Asp Ser Gly Ala ValTrp Lys Gln Ile 355 360 365 Tyr Met Asp Leu Gly Ile Pro Ile Leu Asn SerAla Ala Ser Tyr Asn 370 375 380 Leu Lys Thr Ala Tyr Arg Lys Tyr Leu TyrGly Phe Glu Glu Tyr Cys 385 390 395 400 Arg Ser Ala Asn Ile Gln Phe ArgThr Val His His His Glu Pro Lys 405 410 415 Val Lys Glu Glu Lys Lys AspLeu Glu Glu Ser Met Glu Glu Ala Leu 420 425 430 Lys Leu Asp Gln Glu MetPro Leu Thr Glu Val Lys Ser Glu Pro Glu 435 440 445 Glu Asn Ile Asp SerAsn Ser Glu Ser Glu Arg Glu Glu Ile Glu Leu 450 455 460 Lys Ser Pro ArgGly Arg Arg Arg Ile Ala Arg Asp Val Asn Ser Ile 465 470 475 480 Lys LysGlu Ile Glu Glu Glu Lys Thr Glu Asp Lys Leu Lys Asp Asn 485 490 495 AspThr Glu Asn Lys Asp Val Asp Asp Asp Tyr Glu Thr Ala Glu Lys 500 505 510Lys Glu Asn Glu Leu Leu Leu Gly Arg Lys Asn Thr Pro Lys Gln Lys 515 520525 Glu Lys Lys Ile Lys Lys Gln Glu Asp Ser Asp Lys Asp Ser Asp Glu 530535 540 Glu Glu Glu Lys Ser Gln Glu Arg Glu Glu Thr Glu Ser Lys Cys Asp545 550 555 560 Ser Glu Gly Glu Glu Asp Glu Glu Asp Met Glu Pro Cys LeuThr Gly 565 570 575 Thr Lys Val Lys Val Lys Tyr Gly Arg Gly Lys Thr GlnLys Ile Tyr 580 585 590 Glu Ala Ser Ile Lys Ser Thr Glu Ile Asp Asp GlyGlu Val Leu Tyr 595 600 605 Leu Val His Tyr Tyr Gly Trp Asn Val Ser TyrAsp Glu Trp Val Lys 610 615 620 Ala Asp Arg Ile Ile Trp Pro Leu Asp LysGly Gly Pro Lys Lys Lys 625 630 635 640 Gln Lys Lys Lys Ala Lys Asn LysGlu Asp Ser Glu Lys Asp Glu Lys 645 650 655 Arg Asp Glu Glu Arg Gln LysSer Lys Arg Gly Arg Pro Pro Leu Lys 660 665 670 Ser Thr Leu Ser Ser AsnMet Pro Tyr Gly Leu Ser Lys Thr Ala Asn 675 680 685 Ser Glu Gly Lys SerAsp Ser Cys Ser Ser Asp Ser Glu Thr Glu Asp 690 695 700 Ala Leu Glu LysAsn Leu Ile Asn Glu Glu Leu Ser Leu Lys Asp Glu 705 710 715 720 Leu GluLys Asn Glu Asn Leu Asn Asp Asp Lys Leu Asp Glu Glu Asn 725 730 735 ProLys Ile Ser Ala His Ile Leu Lys Glu Asn Asp Arg Thr Gln Met 740 745 750Gln Pro Leu Glu Thr Leu Lys Leu Glu Val Gly Glu Asn Glu Gln Ile 755 760765 Val Gln Ile Phe Gly Asn Lys Met Glu Lys Ala Glu Glu Val Lys Lys 770775 780 Glu Ala Glu Lys Ser Pro Lys Gly Lys Gly Arg Arg Ser Lys Thr Lys785 790 795 800 Asp Leu Ser Leu Glu Ile Ile Lys Ile Ser Ser Phe Gly GlnAsn Glu 805 810 815 Ala Gly Ser Glu Pro His Ile Glu Ala His Ser Leu GluLeu Ser Ser 820 825 830 Leu Asp Asn Lys Asn Phe Ser Ser Ala Thr Glu AspGlu Ile Asp Gln 835 840 845 Cys Val Lys Glu Lys Lys Leu Lys Arg Lys IleLeu Gly Gln Ser Ser 850 855 860 Pro Glu Lys Lys Ile Arg Ile Glu Asn GlyMet Glu Met Thr Asn Thr 865 870 875 880 Val Ser Gln Glu Arg Thr Ser AspCys Ile Gly Ser Glu Gly Met Lys 885 890 895 Asn Leu Asn Phe Glu Gln HisPhe Glu Arg Glu Asn Glu Gly Met Pro 900 905 910 Ser Leu Ile Ala Glu SerAsn Gln Cys Ile Gln Gln Leu Thr Ser Glu 915 920 925 Arg Phe Asp Ser ProAla Glu Glu Thr Val Asn Ile Pro Leu Lys Glu 930 935 940 Asp Glu Asp AlaMet Pro Leu Ile Gly Pro Glu Thr Leu Val Cys His 945 950 955 960 Glu ValAsp Leu Asp Asp Leu Asp Glu Lys Asp Lys Thr Ser Ile Glu 965 970 975 AspVal Ala Val Glu Ser Ser Glu Ser Asn Ser Leu Val Ser Ile Pro 980 985 990Pro Ala Leu Pro Pro Val Val Gln His Asn Phe Ser Val Ala Ser Pro 995 10001005 Leu Thr Leu Ser Gln Asp Glu Ser Arg Ser Val Lys Glu Ser Asp Ile1010 1015 1020 Thr Ile Glu Val Asp Ser Ile Ala Glu Glu Ser Gln Glu GlyLeu Cys 1025 1030 1035 1040 Glu Arg Glu Ser Ala Asn Gly Phe Glu Thr AsnVal Ala Ser Gly Thr 1045 1050 1055 Cys Ser Ile Ile Val Gln Glu Arg GluSer Arg Glu Lys Gly Gln Lys 1060 1065 1070 Arg Pro Ser Asp Gly Asn SerLeu Met Ala Lys Lys Gln Lys Arg Thr 1075 1080 1085 Pro Lys Arg Thr SerAla Ala Ala Lys Asn Glu Lys Asn Gly Thr Gly 1090 1095 1100 Gln Ser SerAsp Ser Glu Asp Leu Pro Val Leu Asp Asn Ser Ser Lys 1105 1110 1115 1120Cys Thr Pro Val Lys His Leu Asn Val Ser Lys Pro Gln Lys Leu Ala 11251130 1135 Arg Ser Pro Ala Arg Ile Ser Pro His Ile Lys Asp Gly Glu LysAsp 1140 1145 1150 Lys His Arg Glu Lys His Pro Asn Ser Ser Pro Arg ThrTyr Lys Trp 1155 1160 1165 Ser Phe Gln Leu Asn Glu Leu Asp Asn Met AsnSer Thr Glu Arg Ile 1170 1175 1180 Ser Phe Leu Gln Glu Lys Leu Gln GluIle Arg Lys Tyr Tyr Met Ser 1185 1190 1195 1200 Leu Lys Ser Glu Val AlaThr Ile Asp Arg Arg Arg Lys Arg Leu Lys 1205 1210 1215 Lys Lys Asp ArgGlu Val Ser His Ala Gly Ala Ser Met Ser Ser Ala 1220 1225 1230 Ser SerAsp Thr Gly Met Ser Pro Ser Ser Ser Ser Pro Pro Gln Asn 1235 1240 1245Val Leu Ala Val Glu Cys Arg 1250 1255 5 7 PRT Homo sapiens 5 Lys Ala SerIle Phe Leu Lys 1 5 6 10 PRT Homo sapiens 6 Gly Leu Gln Lys Ala Ser IlePhe Leu Lys 1 5 10 7 10 PRT Homo sapiens 7 Lys Ala Ser Ile Phe Leu LysThr Arg Val 1 5 10 8 29 DNA Homo sapiens 8 atggaggagg agaggaatataataccaag 29 9 28 DNA Homo sapiens 9 ctgaaatggt ggtttggaca agcgccga 2810 31 DNA Homo sapiens 10 agagtcacca tgaaggccct tgatgatgag c 31 11 28DNA Homo sapiens 11 tgggattata ttcctctcct cctccatc 28 12 30 DNA Homosapiens 12 atacggcatc aggctttggt gcagtgtcac 30 13 29 DNA Homo sapiens 13agggaatagc tcgccagcag gttttgatg 29 14 30 DNA Homo sapiens 14 tcggcacttgtcatattttc caggtccgac 30 15 5804 DNA Homo sapiens 15 agcgatgtttgcccgtcagt cgagtccgga gtgaggagct cggtcgccga agcggaggga 60 gactcttgagcttcatcttg ccgccgccac ggccaccgcc tggacctttg cccggaggga 120 gctgcagagggtccatcgcc gccgtcctct ggagggcagc gcgattgggg gcccggacct 180 ccagtccgggggggattttt cgtcgtcccc ctccccccaa ccagggagcc cgagcggccg 240 ccaaacaaaggtaccagtcg ccgccgcggg aggaggagga gccggagcct ctgcctcaca 300 gccgctggacccgccgcctt cttccccatc tctcccccgg gcctgctggt tttggggggg 360 agaaggagagaggggactct ggacgtgcca gggtcaaatc tcgcctccga ggaaggtgca 420 actgaacctggtgttttaaa ggataccttg gtcccaaagt catcatgaag gcccttgatg 480 agcctccctatttgacagtg ggcactgatg tgagtgctaa atacagagga gccttttgtg 540 aagccaagatcaagacagca aaaagacttg tcaaagtcaa ggtgacattt agacatgatt 600 cttcaacagtggaagttcag gatgaccaca taaagggccc actaaaggta ggagctattg 660 tggaagtgaagaatcttgat ggtgcatatc aggaagctgt tatcaataaa ctaacagatg 720 cgagttggtacactgtagtt tttgatgacg gagatgagaa gacactgaga cgatcttcac 780 tgtgcctgaaaggagagagg cattttgccg aaagtgaaac attagaccag ctcccactca 840 ccaaccctgagcattttggc actccagtca taggaaagaa aacaaataga ggaagaagat 900 ctaatcatataccagaggaa gagtcttcat catcctccag tgatgaagat gaggatgata 960 ggaaacagattgatgagcta ctaggcaaag ttgtatgtgt agattacatt agtttggata 1020 aaaagaaagcactgtggttt cctgcattgg tggtttgtcc tgattgtagt gatgagattg 1080 ctgtaaaaaaggacaatatt cttgttcgat ctttcaaaga tggaaaattt acttcagttc 1140 caagaaaagatgtccatgaa attactagtg acactgcacc aaagcctgat gctgttttaa 1200 agcaagcctttgaacaggca cttgaatttc acaaaagtag aactattcct gctaactgga 1260 agactgaattgaaagaagat agctctagca gtgaagcaga ggaagaagag gaggaggaag 1320 atgatgaaaaagaaaaggag gataatagca gtgaagaaga agaagaaata gaaccatttc 1380 cagaagaaagggagaacttt cttcagcaat tgtacaaatt tatggaagat agaggtacac 1440 ctattaacaaacaacctgta cttggatatc gaaatttgaa tctctttaag ttattcagac 1500 ttgtacacaaacttggagga tttgataata ttgaaagtgg agctgtttgg aaacaagtct 1560 accaagatcttggaatccct gtcttaaatt cagctgcagg atacaatgtt aaatgtgctt 1620 ataaaaaatacttatatggt tttgaggagt actgtagatc agccaacatt gaatttcaga 1680 tggcattgccagagaaagtt gttaacaagc aatgtaagga gtgtgaaaat gtaaaagaaa 1740 taaaagttaaggaggaaaat gaaacagaga tcaaagaaat aaagatggag gaggagagga 1800 atataataccaagagaagaa aagcctattg aggatgaaat tgaaagaaaa gaaaatatta 1860 agccctctctgggaagtaaa aagaatttat tagaatctat acctacacat tctgatcagg 1920 aaaaagaagttaacattaaa aaaccagaag acaatgaaaa tctggatgac aaagatgatg 1980 acacaactagggtagatgaa tccctcaaca taaaggtaga agctgaggaa gaaaaagcaa 2040 aatctggatacgatgaatgg attaaagcag ataaaatagt aagacctgct gataaaaatg 2100 tgccaaagataaaacatcgg aagaaaataa agaataaatt agacaaagaa aaagacaaag 2160 atgaaaaatactctccaaaa aactgtaaac ttcggcgctt gtccaaacca ccatttcaga 2220 caaatccatctcctgaaatg gtatccaaac tggatctcac tgatgccaaa aactctgata 2280 ctgctcatattaagtccata gaaattactt cgatccttaa tggacttcaa gcttctgaaa 2340 gttctgctgaagacagtgag caggaagatg agagaggtgc tcaagacatg gataataatg 2400 gcaaagaagaatctaagatt gatcatttga ccaacaacag aaatgatctt atttcaaagg 2460 aggaacagaacagttcatct ttgctagaag aaaacaaagt tcatgcagat ttggtaatat 2520 ccaaaccagtgtcaaaatct ccagaaagat taaggaaaga tatagaagta ttatccgaag 2580 atactgattatgaagaagat gaagtcacaa aaaagagaaa ggatgtcaag aaggacacaa 2640 cagataaatcttcaaaacca caaataaaac gtggtaaaag aaggtattgc aatacagaag 2700 agtgtctaaaaactggatca cctggcaaaa aggaagagaa ggccaagaac aaagaatcac 2760 tttgcatggaaaacagtagc aacagctctt cagatgaaga tgaagaagaa acaaaagcaa 2820 agatgacaccaactaagaaa tacaatggtt tggaggaaaa aagaaaatct ctacggacaa 2880 ctggtttctattcaggattt tcagaagtgg cagaaaaaag gattaaactt ttaaataact 2940 ctgatgaaagacttcaaaac agcagggcca aagatcgaaa agatgtctgg tcaagtattc 3000 agggacagtggcctaaaaaa acgctgaaag agcttttttc agactctgat actgaggctg 3060 cagcttccccaccgcatcct gccccagagg agggggtggc agaggagtca ctgcagactg 3120 tggctgaagaggagagttgt tcacccagtg tagaactaga aaaaccacct ccagtcaatg 3180 tcgatagtaaacccattgaa gaagaaacag tagaggtcaa tgacagaaaa gcagaatttc 3240 caagtagtggcagtaattca gtgctaaata cccctcctac tacacctgaa tcgccttcat 3300 cagtcactgtaacagaaggc agccggcagc agtcttctgt aacagtatca gaaccactgg 3360 ctccaaaccaagaagaggtt cgaagtatca agagtgaaac tgatagcaca attgaggtgg 3420 atagtgttgctggggagctc caagacctcc agtctgaagg gaatagctcg ccagcaggtt 3480 ttgatgccagtgtgagctca agcagtagta atcagccaga accagaacat cctgaaaaag 3540 cctgtacaggtcagaaaaga gtgaaagatg ctcagggagg aggaagttca tcaaaaaagc 3600 agaaaagaagccataaagca acagtggtaa acaacaaaaa gaagggaaaa ggcacaaata 3660 gtagtgatagtgaagaactt tcagctggtg aaagtataac taagagtcag ccagtcaaat 3720 cagtttccactggaatgaag tctcatagta ccaaatctcc cgcaaggacg cagtctccag 3780 gaaaatgtggaaagaatggt gataaggatc ctgatctcaa ggaacccagt aatcgattac 3840 ccaaagtttacaaatggagt tttcagatgt cggacctgga aaatatgaca agtgccgaac 3900 gcatcacaattcttcaagaa aaacttcaag aaatcagaaa acattatctg tcattaaaat 3960 ctgaagtagcttccattgat cggaggagaa agcgtttaaa gaagaaagag agagaaagtg 4020 ctgctacatcctcatcctcc tcttcacctt catccagctc cataacagct gctgctatgt 4080 taactttagctgaaccgtca atgtccagcg catcacaaaa tggaatgtca gttgagtgca 4140 ggtgacagcaggacttgcta aggcactttg cacttaatgg ctgttgaggg ccactttttt 4200 tttatactgcacagtggcac aaaaaaatat cagacaagca ctattttata tttaaaaatt 4260 gtttcttgacaagccgactt ggcacttaag tgcacttttg tatgaagaaa agtacaatga 4320 actgcttttcctcaagcaat aattgtttcc aacttgtctg ggaattgtgt gtctggtaac 4380 tggaaggccttccactgtgg caaatggagg cttctcactg cctgtagaga caatacagta 4440 agcatagttaaggggtgggt cagaacatgt taagataact tactgtatat gtattccctt 4500 gtattttgttaaagctggaa catttgatat ttttccattt atttatgaaa aaatatgaac 4560 ctattttcatttgtacaagg taattgtttt ttaaagcaag tcaccttagg gtggctttaa 4620 ttgtataagtcaagcacatg taataaattc aaaacctgca gttaacagga tattagacat 4680 taatcctggtaaccaaatat taaagattct ctttaaaaaa gactgaacat gtttacaggt 4740 ttgaattaggctaaaaggtc ttgcagtggc ttttcatggc ccttcaaatt ggaatggaac 4800 tactgtactttgccattttt ctataaatca gtattttttt ttaattttga tatacattgt 4860 gtgaaaaaagaaaatggcta ataaactgta ttaaatctta aacaatgtat aaagattgta 4920 cttagccagttcaaaggtat atttattcat aatgaattat aacagttata tttttgtgtt 4980 ttcttgtaaatgtttctttt cccttaaata cagataattc atttgtattg cttattttat 5040 tatgagctacaacaaaagga cttcaggaac aagtaatgta ttagtatggt tcaagattgt 5100 tgataggaactgtctcaaaa ggatggtggt tattttaaat ataaatagct aatgggggtg 5160 gtaggcctataaaattaaat gccttgtata agatccaaaa tgaatgcaaa attgttttca 5220 cttgtattgactttatgttg tatgattcca atctctgttc tgtttggcac ttgtatttaa 5280 ttcttcacctttgtaagaca tttgtatatt gtggatgtgt tcattcaagc tatttaatat 5340 ctggcactgttaatacacag tactttattg tacagactgt tttactgttt taattgtagt 5400 tctgtgtactttttttggat ggggctggca tgttttcttt gtttcctggc aatacgacgt 5460 gggaatttcaatgcgttttg ttgtagatgc taacgtgtca gaatccttta cattcaactt 5520 ttctaagaaaagcattttca gtcttgtagt gtgtgcttac agtaactaat tttgttgaaa 5580 atggtttcaagttattcaaa tttgtacagg actgtaaaga tttgttgaca gcaaaatgtt 5640 gaagaaaaaagcttatagaa taaaagctat aaagtatata ttaggatctg caaacaatga 5700 agaattatgtaatatattgt acaaatgtaa agcaaaggct ttgaaataaa atgccattgt 5760 ttgtgaatccttaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 5804 16 1226 PRT Homo sapiens 16Met Lys Ala Leu Asp Glu Pro Pro Tyr Leu Thr Val Gly Thr Asp Val 1 5 1015 Ser Ala Lys Tyr Arg Gly Ala Phe Cys Glu Ala Lys Ile Lys Thr Ala 20 2530 Lys Arg Leu Val Lys Val Lys Val Thr Phe Arg His Asp Ser Ser Thr 35 4045 Val Glu Val Gln Asp Asp His Ile Lys Gly Pro Leu Lys Val Gly Ala 50 5560 Ile Val Glu Val Lys Asn Leu Asp Gly Ala Tyr Gln Glu Ala Val Ile 65 7075 80 Asn Lys Leu Thr Asp Ala Ser Trp Tyr Thr Val Val Phe Asp Asp Gly 8590 95 Asp Glu Lys Thr Leu Arg Arg Ser Ser Leu Cys Leu Lys Gly Glu Arg100 105 110 His Phe Ala Glu Ser Glu Thr Leu Asp Gln Leu Pro Leu Thr AsnPro 115 120 125 Glu His Phe Gly Thr Pro Val Ile Gly Lys Lys Thr Asn ArgGly Arg 130 135 140 Arg Ser Asn His Ile Pro Glu Glu Glu Ser Ser Ser SerSer Ser Asp 145 150 155 160 Glu Asp Glu Asp Asp Arg Lys Gln Ile Asp GluLeu Leu Gly Lys Val 165 170 175 Val Cys Val Asp Tyr Ile Ser Leu Asp LysLys Lys Ala Leu Trp Phe 180 185 190 Pro Ala Leu Val Val Cys Pro Asp CysSer Asp Glu Ile Ala Val Lys 195 200 205 Lys Asp Asn Ile Leu Val Arg SerPhe Lys Asp Gly Lys Phe Thr Ser 210 215 220 Val Pro Arg Lys Asp Val HisGlu Ile Thr Ser Asp Thr Ala Pro Lys 225 230 235 240 Pro Asp Ala Val LeuLys Gln Ala Phe Glu Gln Ala Leu Glu Phe His 245 250 255 Lys Ser Arg ThrIle Pro Ala Asn Trp Lys Thr Glu Leu Lys Glu Asp 260 265 270 Ser Ser SerSer Glu Ala Glu Glu Glu Glu Glu Glu Glu Asp Asp Glu 275 280 285 Lys GluLys Glu Asp Asn Ser Ser Glu Glu Glu Glu Glu Ile Glu Pro 290 295 300 PhePro Glu Glu Arg Glu Asn Phe Leu Gln Gln Leu Tyr Lys Phe Met 305 310 315320 Glu Asp Arg Gly Thr Pro Ile Asn Lys Gln Pro Val Leu Gly Tyr Arg 325330 335 Asn Leu Asn Leu Phe Lys Leu Phe Arg Leu Val His Lys Leu Gly Gly340 345 350 Phe Asp Asn Ile Glu Ser Gly Ala Val Trp Lys Gln Val Tyr GlnAsp 355 360 365 Leu Gly Ile Pro Val Leu Asn Ser Ala Ala Gly Tyr Asn ValLys Cys 370 375 380 Ala Tyr Lys Lys Tyr Leu Tyr Gly Phe Glu Glu Tyr CysArg Ser Ala 385 390 395 400 Asn Ile Glu Phe Gln Met Ala Leu Pro Glu LysVal Val Asn Lys Gln 405 410 415 Cys Lys Glu Cys Glu Asn Val Lys Glu IleLys Val Lys Glu Glu Asn 420 425 430 Glu Thr Glu Ile Lys Glu Ile Lys MetGlu Glu Glu Arg Asn Ile Ile 435 440 445 Pro Arg Glu Glu Lys Pro Ile GluAsp Glu Ile Glu Arg Lys Glu Asn 450 455 460 Ile Lys Pro Ser Leu Gly SerLys Lys Asn Leu Leu Glu Ser Ile Pro 465 470 475 480 Thr His Ser Asp GlnGlu Lys Glu Val Asn Ile Lys Lys Pro Glu Asp 485 490 495 Asn Glu Asn LeuAsp Asp Lys Asp Asp Asp Thr Thr Arg Val Asp Glu 500 505 510 Ser Leu AsnIle Lys Val Glu Ala Glu Glu Glu Lys Ala Lys Ser Gly 515 520 525 Tyr AspGlu Trp Ile Lys Ala Asp Lys Ile Val Arg Pro Ala Asp Lys 530 535 540 AsnVal Pro Lys Ile Lys His Arg Lys Lys Ile Lys Asn Lys Leu Asp 545 550 555560 Lys Glu Lys Asp Lys Asp Glu Lys Tyr Ser Pro Lys Asn Cys Lys Leu 565570 575 Arg Arg Leu Ser Lys Pro Pro Phe Gln Thr Asn Pro Ser Pro Glu Met580 585 590 Val Ser Lys Leu Asp Leu Thr Asp Ala Lys Asn Ser Asp Thr AlaHis 595 600 605 Ile Lys Ser Ile Glu Ile Thr Ser Ile Leu Asn Gly Leu GlnAla Ser 610 615 620 Glu Ser Ser Ala Glu Asp Ser Glu Gln Glu Asp Glu ArgGly Ala Gln 625 630 635 640 Asp Met Asp Asn Asn Gly Lys Glu Glu Ser LysIle Asp His Leu Thr 645 650 655 Asn Asn Arg Asn Asp Leu Ile Ser Lys GluGlu Gln Asn Ser Ser Ser 660 665 670 Leu Leu Glu Glu Asn Lys Val His AlaAsp Leu Val Ile Ser Lys Pro 675 680 685 Val Ser Lys Ser Pro Glu Arg LeuArg Lys Asp Ile Glu Val Leu Ser 690 695 700 Glu Asp Thr Asp Tyr Glu GluAsp Glu Val Thr Lys Lys Arg Lys Asp 705 710 715 720 Val Lys Lys Asp ThrThr Asp Lys Ser Ser Lys Pro Gln Ile Lys Arg 725 730 735 Gly Lys Arg ArgTyr Cys Asn Thr Glu Glu Cys Leu Lys Thr Gly Ser 740 745 750 Pro Gly LysLys Glu Glu Lys Ala Lys Asn Lys Glu Ser Leu Cys Met 755 760 765 Glu AsnSer Ser Asn Ser Ser Ser Asp Glu Asp Glu Glu Glu Thr Lys 770 775 780 AlaLys Met Thr Pro Thr Lys Lys Tyr Asn Gly Leu Glu Glu Lys Arg 785 790 795800 Lys Ser Leu Arg Thr Thr Gly Phe Tyr Ser Gly Phe Ser Glu Val Ala 805810 815 Glu Lys Arg Ile Lys Leu Leu Asn Asn Ser Asp Glu Arg Leu Gln Asn820 825 830 Ser Arg Ala Lys Asp Arg Lys Asp Val Trp Ser Ser Ile Gln GlyGln 835 840 845 Trp Pro Lys Lys Thr Leu Lys Glu Leu Phe Ser Asp Ser AspThr Glu 850 855 860 Ala Ala Ala Ser Pro Pro His Pro Ala Pro Glu Glu GlyVal Ala Glu 865 870 875 880 Glu Ser Leu Gln Thr Val Ala Glu Glu Glu SerCys Ser Pro Ser Val 885 890 895 Glu Leu Glu Lys Pro Pro Pro Val Asn ValAsp Ser Lys Pro Ile Glu 900 905 910 Glu Glu Thr Val Glu Val Asn Asp ArgLys Ala Glu Phe Pro Ser Ser 915 920 925 Gly Ser Asn Ser Val Leu Asn ThrPro Pro Thr Thr Pro Glu Ser Pro 930 935 940 Ser Ser Val Thr Val Thr GluGly Ser Arg Gln Gln Ser Ser Val Thr 945 950 955 960 Val Ser Glu Pro LeuAla Pro Asn Gln Glu Glu Val Arg Ser Ile Lys 965 970 975 Ser Glu Thr AspSer Thr Ile Glu Val Asp Ser Val Ala Gly Glu Leu 980 985 990 Gln Asp LeuGln Ser Glu Gly Asn Ser Ser Pro Ala Gly Phe Asp Ala 995 1000 1005 SerVal Ser Ser Ser Ser Ser Asn Gln Pro Glu Pro Glu His Pro Glu 1010 10151020 Lys Ala Cys Thr Gly Gln Lys Arg Val Lys Asp Ala Gln Gly Gly Gly1025 1030 1035 1040 Ser Ser Ser Lys Lys Gln Lys Arg Ser His Lys Ala ThrVal Val Asn 1045 1050 1055 Asn Lys Lys Lys Gly Lys Gly Thr Asn Ser SerAsp Ser Glu Glu Leu 1060 1065 1070 Ser Ala Gly Glu Ser Ile Thr Lys SerGln Pro Val Lys Ser Val Ser 1075 1080 1085 Thr Gly Met Lys Ser His SerThr Lys Ser Pro Ala Arg Thr Gln Ser 1090 1095 1100 Pro Gly Lys Cys GlyLys Asn Gly Asp Lys Asp Pro Asp Leu Lys Glu 1105 1110 1115 1120 Pro SerAsn Arg Leu Pro Lys Val Tyr Lys Trp Ser Phe Gln Met Ser 1125 1130 1135Asp Leu Glu Asn Met Thr Ser Ala Glu Arg Ile Thr Ile Leu Gln Glu 11401145 1150 Lys Leu Gln Glu Ile Arg Lys His Tyr Leu Ser Leu Lys Ser GluVal 1155 1160 1165 Ala Ser Ile Asp Arg Arg Arg Lys Arg Leu Lys Lys LysGlu Arg Glu 1170 1175 1180 Ser Ala Ala Thr Ser Ser Ser Ser Ser Ser ProSer Ser Ser Ser Ile 1185 1190 1195 1200 Thr Ala Ala Ala Met Leu Thr LeuAla Glu Pro Ser Met Ser Ser Ala 1205 1210 1215 Ser Gln Asn Gly Met SerVal Glu Cys Arg 1220 1225 17 23 DNA Homo sapiens 17 tgacggatcctgcggccgca aag 23 18 27 DNA Homo sapiens 18 tctggaattc cttcccagagagagggc 27 19 25 DNA Homo sapiens 19 tctggaattc attcttttta cttcc 25 2029 DNA Homo sapiens 20 tctggaattc cctgatcaga atgtgtagg 29 21 30 DNA Homosapiens 21 tctggaattc cccagatttt cattgtcttc 30 22 27 DNA Homo sapiens 22tctggaattc cctaccctag ttgtgtc 27 23 27 DNA Homo sapiens 23 tctggaattcgctttaatcc attcatc 27 24 27 DNA Homo sapiens 24 tctggaattc gctttttcttcctcagc 27 25 10 PRT Homo sapiens 25 Ile Lys Pro Ser Leu Gly Ser Lys LysAsn 1 5 10 26 9 PRT Homo sapiens 26 Lys Pro Ser Leu Gly Ser Lys Lys Asn1 5 27 8 PRT Homo sapiens 27 Ile Lys Pro Ser Leu Gly Ser Lys 1 5 28 7PRT Homo sapiens 28 Lys Pro Ser Leu Gly Ser Lys 1 5 29 7 PRT Homosapiens 29 Pro Ser Leu Gly Ser Lys Lys 1 5 30 7 PRT Homo sapiens 30 IleLys Pro Ser Leu Gly Ser 1 5 31 7 PRT Homo sapiens 31 Ser Leu Gly Ser LysLys Asn 1 5 32 8 PRT Homo sapiens 32 Lys Pro Ser Leu Gly Ser Lys Lys 1 533 8 PRT Homo sapiens 33 Pro Ser Leu Gly Ser Lys Lys Asn 1 5

We claim:
 1. An isolated polynucleotide that encodes a polypeptidecomprising SEQ ID NO:3 or a variant thereof.
 2. The polynucleotide ofclaim 1, wherein the variant specifically binds to an antibody raisedagainst the polypeptide comprising SEQ ID NO:3 or an antibody obtainedfrom a subject having cancer.
 3. The polynucleotide of claim 1, whereinthe polynucleotide comprises a SEQ ID NO:1 or a fragment or variantthereof.
 4. The polynucleotide of claim 1, wherein the polynucleotideconsists of SEQ ID NO:1 or a fragment thereof.
 5. The polynucleotide ofclaim 1, wherein the polypeptide comprises SEQ ID NO:2 or a fragment orvariant thereof.
 6. The polynucleotide of claim 1, wherein thepolypeptide consists of SEQ ID NO:3.
 7. The polynucleotide of claim 1,wherein the polypeptide consists of SEQ ID NO:2.
 8. The polynucleotideof claim 1, wherein the polynucleotide sequence is at least about 80%identical to SEQ ID NO:1.
 9. The polynucleotide of claim 1, wherein thepolynucleotide sequence is at least about 90% identical to SEQ ID NO:1.10. The polynucleotide of claim 1, wherein the polypeptide is at leastabout 80% identical to SEQ ID NO:2 or SEQ ID NO:3.
 11. Thepolynucleotide of claim 1, wherein the polypeptide is at least about 90%identical to SEQ ID NO:2 or SEQ ID NO:3.
 12. A purified polypeptideencoded by the polynucleotide of claim
 1. 13. The polypeptide of claim12, wherein the polypeptide comprises SEQ ID NO:2 or SEQ ID NO:3. 14.The polypeptide of claim 12, wherein the polypeptide consists of SEQ IDNO:2 or SEQ ID NO:3.
 15. The polypeptide of claim 12, wherein thepolypeptide is at least about 80% identical to SEQ ID NO:2 or SEQ IDNO:3.
 16. The polypeptide of claim 12, wherein the polypeptide is atleast about 90% identical to SEQ ID NO:2 or SEQ ID NO:3.
 17. A vectorcomprising the polynucleotide of claim
 1. 18. A host comprising thepolynucleotide of claim 1 or a vector comprising the polynucleotide ofclaim
 1. 17. A fusion protein comprising the polypeptide of claim 13.18. An antibody or fragment thereof that specifically binds thepolypeptide of claim
 12. 19. The antibody or fragment of claim 18,wherein the antibody was raised against a polypeptide comprising SEQ IDNO:2 or SEQ ID NO:3.
 20. A pharmaceutical composition comprising thepolypeptide of claim 12 and a pharmaceutically acceptable vehicle.
 21. Apharmaceutical composition comprising the antibody or fragment of claim18 and a pharmaceutically acceptable vehicle.
 22. An immunogeniccomposition comprising the polypeptide of claim
 12. 23. A vaccinecomprising the polypeptide of claim
 12. 24. A method of inducing animmune response against a cancer or a tumor in a subject which comprisesadministering to the subject at least one polypeptide of claim
 12. 25.The method of claim 24, wherein the cancer is breast, lung, colon,pancreas, or ovarian cancer.
 26. A method of treating, preventing, orinhibiting a cancer or a tumor in a subject which comprisesadministering to the subject at least one polypeptide of claim 12, atleast one antibody raised against the polypeptide of claim 12, or both.27. The method of claim 26, wherein the cancer is breast, lung, colon,pancreas, or ovarian cancer.
 28. A marker for diagnosing a cancer orsusceptibility of obtaining the cancer in a subject comprising thepolynucleotide of claim
 1. 29. The marker of claim 28, wherein thecancer is breast, lung, colon, pancreas, or ovarian cancer.
 30. A markerfor diagnosing a cancer or susceptibility of obtaining the cancer in asubject comprising the polypeptide of claim
 12. 31. A method fordetecting the presence of a cancer in a subject which comprises: (a)obtaining a biological sample from the subject; (b) contacting thebiological sample with a binding agent that binds to the polypeptide ofclaim 12; (c) detecting in the sample an amount of polypeptide thatbinds to the binding agent; and (d) comparing the amount of polypeptideto a control and therefrom determining the presence of a cancer in thesubject.
 32. A method for detecting the presence of a cancer in asubject which comprises: (a) obtaining a biological sample from thepatient; (b) contacting the biological sample with the polynucleotide ofclaim 1; (c) detecting in the sample an amount of a secondpolynucleotide that hybridizes to the polynucleotide of claim 1; and (d)comparing the amount of the second polynucleotide that hybridizes to thepolynucleotide of claim 1 to a control, and therefrom determining thepresence of the cancer in the patient.
 33. A diagnostic kit comprisingat least one reagent selection from the group consisting of: (a) apolynucleotide that encodes a polypeptide comprising SEQ ID NO:3 or avariant thereof; (b) the polypeptide comprising SEQ ID NO:3 or a variantthereof; (c) a polypeptide comprising SEQ ID NO:2 or a fragment orvariant thereof; or (d) an antibody that specifically binds thepolypeptide of (b) or (c); and instructions for use.
 34. A method forinhibiting the development of a cancer in a subject which comprises: (a)incubating CD4+ T cells, CD8+ T cells, or both with at least onecomponent selected from the group consisting of: (i) at least onepolynucleotide that encodes a polypeptide comprising SEQ ID NO:3 or avariant thereof; (ii) the polypeptide comprising SEQ ID NO:3 or avariant thereof; (iii) at least one polypeptide comprising SEQ ID NO:2or a fragment or variant thereof; and (iv) antigen presenting cells thatexpress the polypeptide of (ii) or (iii), such that T cell proliferate;and (b) administering to the subject an effective amount of theproliferated T cells, and thereby inhibiting the development of a cancerin the subject.